Transformation system in camelina sativa

ABSTRACT

The present invention relates to plant biotechnology and specifically to a method for genetically transforming  Camelina sativa  with Agrobacterium-mediated transformation system. It comprises  Camelina sativa  for producing homologous and heterologous recombinant products including oil and protein products and assessing and screening the efficacy of plant transformation. Also disclosed are transgenic  Camelina sativa  plants, seeds as well as cells, cell-lines and tissue of  Camelina sativa.

TECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates to plant biotechnology and plantcell transformation. More particularly the invention relates to a methodfor genetically transforming Camelina sativa usingAgrobacterium-mediated transformation of a plant tissue explant andsubsequent regeneration of the transformed cells into whole Camelinasativa plants. It further relates to the use of anAgrobacterium-mediated transformation method of Camelina sativa forproducing homologous or heterologous recombinant products including forexample proteins, enzymes and oil products and for assessing andscreening the properties, and effects of DNA sequences and recombinantDNA constructs in plants.

BACKGROUND OF THE INVENTION

[0002] Genetic transformation of plants allows the introduction of genesof any origin into the target species providing novel products for e.g.agricultural, horticultural, nutritional and chemical applications.Furthermore, transgenic plants provide more information about basicplant biology, gene function and regulation. In many plant species,traditional plant breeding is limited due to the fact that the existinggene pool is narrow and prevents further development. Alteration ofsingle characteristics can be time-consuming and even impossible withoutchanging any other properties. Major applications of genetictransformation focus on the improvement of for example diseaseresistance, insect resistance, herbicide tolerance, modified qualitycharacteristics such as modification of oil and protein compositions aswell as on improving stress tolerance and modifying growthcharacteristics. In other applications transgenic plants are used asbioreactors for producing foreign proteins or plant secondarymetabolites.

[0003] Several vector systems have been developed to be used in higherplants for transferring genes into plant tissue e.g. the use of plantviruses as vectors, direct gene transfer using DNA fragments notattached to a vector and Agrobacterium-mediated gene transformation.

[0004] Agrobacterium-mediated gene transformation is the most widelyused method to transfer genes in plants using either Agrobacteriumtumefaciens or Agrobacterium rhizogenes. Several Agrobacterium-mediatedsystems and methods for transforming plants and plant cells have beendisclosed for example in WO 84/02920, EP 289478, U.S. Pat. No.5,352,605, U.S. Pat. No. 5,378,619, U.S. Pat. No. 5,416,011, U.S. Pat.No. 5,569,834, U.S. Pat. No. 5,959,179, U.S. Pat. No. 6,018,100, and

TECHNICAL FIELD OF THE INVENTION

[0005] The present invention relates to plant biotechnology and plantcell transformation. More particularly the invention relates to a methodfor genetically transforming Camelina sativa usingAgrobacterium-mediated transformation of a plant tissue explant andsubsequent regeneration of the transformed cells into whole Camelinasativa plants. It further relates to the use of anAgrobacterium-mediated transformation method of Camelina sativa forproducing homologous or heterologous recombinant products including forexample proteins, enzymes and oil products and for assessing andscreening the properties, and effects of DNA sequences and recombinantDNA constructs in plants.

BACKGROUND OF THE INVENTION

[0006] Genetic transformation of plants allows the introduction of genesof any origin into the target species providing novel products for e.g.agricultural, horticultural, nutritional and chemical applications.Furthermore, transgenic plants provide more information about basicplant biology, gene function and regulation. In many plant species,traditional plant breeding is limited due to the fact that the existinggene pool is narrow and prevents further development. Alteration ofsingle characteristics can be time-consuming and even impossible withoutchanging any other properties. Major applications of genetictransformation focus on the improvement of for example diseaseresistance, insect resistance, herbicide tolerance, modified qualitycharacteristics such as modification of oil and protein compositions aswell as on improving stress tolerance and modifying growthcharacteristics. In other applications transgenic plants are used asbioreactors for producing foreign proteins or plant secondarymetabolites.

[0007] Several vector systems have been developed to be used in higherplants for transferring genes into plant tissue e.g. the use of plantviruses as vectors, direct gene transfer using DNA fragments notattached to a vector and Agrobacterium-mediated gene transformation.

[0008] Agrobacterium-mediated gene transformation is the most widelyused method to transfer genes in plants using either Agrobacteriumtumefaciens or Agrobacterium rhizogenes. Several Agrobacterium-mediatedsystems and methods for transforming plants and plant cells have beendisclosed for example in WO 84/02920, EP 289478, U.S. Pat. No.5,352,605, U.S. Pat. No. 5,378,619, U.S. Pat. No. 5,416,011, U.S. Pat.No. 5,569,834, U.S. Pat. No. 5,959,179, U.S. Pat. No. 6,018,100, and

[0009] Many of said methods are especially applied for oil crops such asBrassicaceae including Brassica rapa ssp. oleifera (Radke et al., PlantCell Rep. 11:499-505, 1992) and Brassica campestris (Kuvshinov et al.Plant Cell Rep. 18:773-777, 1999). Patents U.S. Pat. No. 5,188,958, U.S.Pat. No. 5,463,174 and U.S. Pat. No. 5,750,871 disclose thetransformation of Brassica species using Agrobacterium-mediatedtransformation system.

[0010] Several transformation strategies utilizing theAgrobacterium-mediated transformation system have been developed. Thebinary vector strategy is based on a two-plasmid system where T-DNA isin a different plasmid from the rest of the Ti plasmid. In thecointegration strategy a small portion of the T-DNA is placed in thesame vector as the foreign gene, which vector subsequently recombineswith the Ti plasmid.

[0011] The production of transgenic plants has become routine for manyplant species, but no universal transformation method for differentplant species exists, since transformation and regeneration capacityvaries among species and even with different explants. However, there isa need for developing alternative transformation systems and methodsespecially in oil crop.

[0012]Camelina sativa (gold of pleasure or false flax), one of the mostimportant oil crops in Europe during bronze and iron age, has been grownin Europe for centuries. It was especially used as lamp oil, but also inedible products. Oil products obtained from Camelina sativa have beenused for producing food spreads as described in the patent U.S. Pat. No.6,117,476.

[0013]Camelina sativa (L. Crantz) belongs to the family Brassicaceae inthe tribe Sisymbrieae and both spring- and winter forms are inproduction. It is a low-input crop adapted to low fertility soils.Results from long-term experiments in Central Europe have shown that theseed yields of Camelina sativa are comparable to the yields of oil seedrape.

[0014] Due to the high oil content of Camelina sativa seeds varyingabout 30-40%, there has been a renewed interest in Camelina sativa oil.Camelina sativa seeds have a high content of polyunsaturated fattyacids, about 50-60% with an excellent balance of useful fatty acidsincluding 30-40% of alpha-linolenic acid, which is an omega-3 oil.Omega-3 oils resemble marine oils and are rarely found in other oilcrops. Furthermore, Camelina sativa seed contains a high amount oftocopherols (appr. 600 ppm) with a unique oxidative stability. Moreover,the oil is low in glucosinolates (Matthäus and Zubr, Industrial Cropsand Products 12:9-18, 2000). A quality problem for food and feed uses ofCamelina sativa is that it contains relatively high amount of erucicacid (2-4%) and 11-eicosenoic acid (gondoic acid). Erucic acid is poorlydigested and causes myocardial lesions in animals. Said problem causingerucic and 11-eicosenoic acids can be removed from the oil and used forother non-nutritional applications, which include the use of high-eruricacid containing oils as lubricants. Industrial applications mightrequire prominence of such fatty acid of singular importance.

[0015] As Camelina sativa is a minor crop species, very little has beendone in terms of its breeding aside from testing different accessionsfor agronomic traits and oil profiles. A mutation breeding experiment toinduce variation in the fatty acid profiles has reported three to fourfold differences (Buchsenschutz-Northdurft et al., 3rd EuropeanSymposium on Industrial Crops and Products, France, 1996). Applicationof tissue culture techniques to Camelina sativa are restricted to twoapproaches. Camelina sativa has been used in a somatic fusion with otherBrassica species (Narasimhulu et al., Plant Cell Rep. 13:657-660, 1994;Hansen, Crucifer. News 19:55-56, 1997; Sigareva and Earle, Theor. Appl.Genet. 98:164-170, 1999) and regenerated interspecific hybrid plantswere obtained (Sigareva and Earle, Theor. Appl. Genet. 98:164-170,1999). Recently, Camelina sativa shoots have been regenerated from leafexplants (Tattersall and Millam, Plant Cell Tissue and Organ Culture55:147-149, 1999).

[0016] Present invention provides a genetic transformation system forCamelina sativa, which would address rapid improvement of this crop fordifferent end-uses, which include the production of homologous andheterologous recombinant DNA products. Examples of homologousrecombinant products comprise e.g. unique protein or oil products whichare specific for Camelina sativa, whereas heterologous products includeforeign proteins, enzymes, etc.

[0017] Another embodiment of the present invention is to provide a novelmodel plant for replacing e.g. Arabidopsis and tobacco.

[0018] A further embodiment is to provide transgenic Camelina sativaplants, plant tissue, plant cells and cell lines and seed.

SUMMARY OF THE INVENTION

[0019] The objectives of the present invention are achieved by themethod of the present invention, which enables the use ofAgrobacterium-mediated transformation method for genetic transformationof Camelina sativa explants.

[0020] The specific advantage of the present method is that Camelinasativa has characteristics which make it suitable for use in efficientgenetic transformation and subsequent production of heterologous andhomologous gene products. Camelina sativa germinates and grows rapidlyand already after 10 days from germination explants can be excised fromplantlets. Genetically transformed Camelina sativa plants can betransfered to greenhouse after four weeks from transformation event. Thetransformation efficiency of Camelina sativa is high compared to otherplants including Brassica species. The rapid growth of Camelina sativaenables that the transformation method can be scaled up for futureapplications.

[0021] Transformation of Camelina sativa is effective even withoutselection avoiding the use of a selectable marker gene, which makes thetransformation of Camelina sativa attractive for applications, sincepossible harmful effects of the marker genes used in cloning vectors isa concern in genetically engineered plants.

[0022] The present invention provides a novel method to geneticallytransform Camelina sativa using Agrobacterium-mediated transformation.The method and the products and means utilized in said method are asdefined in the claims of the present invention and they provide anefficient, reliable and convenient transformation system for producingCamelina sativa crop with improved properties using transgenicimprovement and recombinant DNA technologies.

[0023] The present invention is related to transgenic Camelina sativaplants obtainable with the method of the present invention as defined inthe claims by optionally sterilizing one or more seeds of Camelinasativa and germinating and growing said seeds into plants, providingexplants of Camelina sativa plants, contacting the explants of Camelinasativa with an Agrobacterium vector comprising at least one recombinantDNA construct, an optional selectable marker gene and an optionalenhancer, allowing the transformation to take place on a cell culturemedium optionally supplemented with at least one hormone and/or growthfactor, selecting the transformed tissue of Camelina sativa on a mediumoptionally containing at least one selective component, inducing theregeneration of one or more shoots from the transformed explants on acell culture medium optionally containing at least one hormone and/orgrowth factor and growing the shoots into whole Camelina sativa plants.

[0024] The invention is also related to transgenic Camelina sativa planttissue obtainable with the method defined in the claims.

[0025] The invention further relates to transgenic Camelina sativa plantcells or cell lines obtainable with the method defined in the claims.

[0026] The invention is also related to transgenic Camelina sativa seedobtainable with the method defined in the claims.

A SHORT DESCRIPTION OF THE DRAWINGS

[0027]FIG. 1 shows in vitro cultured Camelina sativa plant.

[0028]FIG. 2 shows regenerated shoots of Camelina sativa on leaf segmentexplants.

[0029]FIG. 3a depicts GUS expression in callus tissue of Camelinasativa. The arrowheads point to GUS stained inclusions.

[0030]FIG. 3b depicts GUS expression in callus tissue of Camelinasativa. The arrowheads point to GUS stained inclusions.

[0031]FIG. 4 shows results of PCR amplification of a transgenicinsertion. The PCR was carried out using specific primers developed forthe central part of uidA gene. The length of the DNA sequence betweenthe primers is about 700 nucleotides and thus the size of theamplification product is also 700 nucleotides. Samples on the gel aremarked as follows: NT, non-transgenic Camelina sativa (negativecontrol); T, transgenic GUS positive line of Camelina sativa expressinguidA gene; +, uidA gene sequence cloned in pBluescript vector aspositive control. M is a one kilobase (1 kb) marker ladder (Fermentas).The sizes of some of the marker bands are shown on the left side of thefigure. No PCR product was obtained when non-transgenic Camelina sativaDNA was used as template, whereas when using transgenic Camelina sativaan amplification product of 700 nucleotides corresponding to thepositive control was obtained.

[0032]FIG. 5 shows Camelina sativa plantlets grown in greenhouseconditions. The plantlets are transgenic shoots recovered and rootedafter in vitro selection of transformed explants of Camelina sativa.

DETAILED DESCRIPTION OF THE INVENTION

[0033] In the present invention the terms used have the meaning theygenerally have in the fields of conventional plant breeding, plantbiochemistry and production of transgenic plants, including recombinantDNA technology as well as agriculture and horticulture. Some terms,however, are used with a somewhat deviating or broader meaning in thiscontext. Accordingly, in order to avoid uncertainty caused by terms withunclear meaning some of the terms used in this specification and in theclaims are defined in more detail below.

[0034] Abbreviations

[0035] BAP 6-benzylaminopurine

[0036] 2,4-D 2,4-dichlorophenoxyacetic acid

[0037] GUS β-glucuronidase (uidA reporter gene)

[0038] hpt gene encoding for hygromycin phosphotransferase

[0039] Hyg hygromycin

[0040] IAA indole-3-acetic acid

[0041] Kan kanamycin

[0042] MS Murashige and Skoog medium

[0043] NAA α-naphthaleneacetic acid

[0044] nptII gene encoding for neomycin phosphotransferase II

[0045] Rif rifampicin

[0046] Spe spectinomycin

[0047] Str streptomycin

[0048] Tc ticarcillin

[0049] uidA gene encoding for β-glucuronidase (GUS)

[0050] YEB medium for cultivation of Agrobacterium cells

[0051] Definitions

[0052] “Camelina sativa” belongs to the family of Brassicaceae in thetribe Sisymbriae. The seed yields of Camelina sativa are comparable tothe seed yields of oil seed rape. Useful varieties of Camelina sativaare for example var. Calina and var. Calinca which are hereby mentionedbut not claimed.

[0053] The term “Agrobacterium” means Agrobacterium tumefaciens,Agrobacterium rhizogenes or another Agrobacterium species useful forgenetic transformation of plants to produce genetically modified plants.

[0054] “Agrobacterium tumefaciens” is a naturally occuring bacteriumwhich when containing a circular Ti (Tumor inducing) plasmid is able toform crown gall disease in many species of dicotyledonous plants. Crowngall occurs when a wound is invaded by Agrobacterium. Agrobacteriumtumefaciens natively has the ability to transfer a portion of its DNAcalled T-DNA, into the genome of the plant cells. InAgrobacterium-mediated transformation T-DNA is replaced with a foreignset of genes, thus, making the bacterium capable of transferring theforeign genes into the genome of the plant cell. “Agrobacteriumtumefaciens” can be one of the three different strains of Agrobacteriumused in the transformation of Camelina sativa or an equivalent strainsuitable for the transformation. The strain LB4404 carries the plasmidpAL4404, the strain C58C1 carries the plasmid pGV3850 and the strainEHA105 carries the plasmid pTiBo542.

[0055] The term “Agrobacterium-mediated genetic transformation” in thepresent invention means that Agrobacterium is used as a vector which isable to transfer foreign gene(s) to Camelina sativa cells. The T-DNAportion of the Ti plasmid is replaced with the foreign gene and, afterthe Agrobacterium infection, transferred into the plant chromosomal DNA.

[0056] The term “transgenic plant” means a plant, especially Camelinasativa plant, which is obtained using the method disclosed in thepresent invention. The optionally sterilized seeds of Camelina sativaare germinated and grown to plants, which provide explants for use inAgrobacterium-mediated transformation method. An Agrobacterium straincontaining an Agrobacterium vector comprising at least one recombinantDNA construct and an optional selectable marker gene is allowed totransform Camelina sativa cells in a cell culture medium optionallysupplemented with at least one hormone and/or growth factor. Selectionon a medium optionally containing at least one selective component isfollowed by the regeneration of one or more shoots or roots from thetransformed explants on a cell culture medium optionally containing atleast one hormone and/or growth factor. The shoots are grown into wholetransgenic Camelina sativa plants.

[0057] The term “explant” means a part or a piece which is taken from aplant, in this this context from Camelina sativa. These pieces or tissueexplants can be excised from hypocotyl, cotyledon, stem, leaf or otherplant organs and can be used for in vitro culture and for transformationexperiments. The term “in vitro explant” means a Camelina sativa explantexcised from hypocotyl, cotyledon, stem, leaf or other plant organsoriginating from plants grown in vitro preferably under sterileconditions on culture media. An “explant” or “in vitro explant” can be aleaf segment.

[0058] The term “leaf segment” means a piece of leaf from preferably invitro grown Camelina sativa. The leaves of in vitro grown Camelinasativa can be rather small in size for example 2-4 cm long and 0.5-1 cmwide. Accordingly, narrow leaves are cut across the leaf while largerleaves are also cut in half along the central vein.

[0059] The term “recombinant DNA construct” means a DNA sequenceincluding linear or circular vector, plasmid or insert created byligating or joining together pieces of DNA that are not normallycontiguous in nature. The construct, which is transfered into the plantcell, comprises a specific gene of interest, which is desired to beintroduced into the germline of the plant, and an optional selectablemarker gene that confers upon the plant cell a resistance to a chemicalselective component (selection agent).

[0060] The term “selectable marker gene” means an optionally used genein plant transformation such as the gene for neomycin phosphotransferase(npt II), which expresses an enzyme conferring resistance to theantibiotic kanamycin and the related antibiotics neomycin, paromomycin,gentamicin, and G418, or the gene for hygromycin phosphotransferase(hpt), which expresses an enzyme conferring resistance to hygromycin.Other selectable marker genes include genes encoding herbicideresistance, metal resistance or sensitivity, for example Cu-resistanceor sensitivity, genes utilizing special carbohydrate sources or othermetabolites including for example mannose or other selection systems.

[0061] The term “selection of the transformed tissue of Camelina sativa”means that the transformed tissue is grown on a medium containing asubstrate allowing the selection of transformed tissues which carry amarker gene. Preferred selective substances are antibiotics, for examplehygromycin or kanamycin, but other selection systems such as herbicides,metal resistance or sensitivity, including e.g. Cu-resistance orsensitivity and special carbohydrate sources or other metabolites areapplicable. The use of a marker gene e.g. hpt or nptII is optional,since possible harmful effects of the marker genes used with plantcloning vectors is one area of concern with genetically engineeredplants. Antibiotic selection begins preferably immediately aftertransformation and the result of the selection can be seen in 1-2 weeks,when the explants begin to form callus.

[0062] The term “Murashige and Skoog (MS) medium or equivalent” or “MSmedium or equivalent” means that preferably Murashige and Skoog's growthmedium, that is MS medium, is used in the method of the presentinvention (Murashige and Skoog, Physiol Plant. 15:472-493, 1962). Anyother medium suitable for the purposes of the present invention can alsobe used including for example the B5 medium. The growth medium can be inliquid form or made solid or semisolid using an appropriate amount ofagar or gelrite for example 0.7 g/l. The concentration of the medium canoptionally varies from half strength to 2× strength.

[0063] The term “inoculation with Agrobacterium” means that the Camelinasativa explants are inoculated with bacteria by placing them inAgrobacterium suspension to enable the transformation of Camelina sativawith Agrobacterium. Before inoculation an overnight culture ofAgrobacterium has been diluted in a Murashige and Skoog (MS) solution oranother suitable growth medium to enable the transformation of Camelinasativa with Agrobacterium.

[0064] The term “co-cultivation” means that Camelina sativa explants areplaced preferably on solid Murashige and Skoog (MS) agar medium or anequivalent medium supplemented with at least one hormone, such ascytokinin or auxin, and optionally with acetosyringone forco-cultivation. Explants are co-cultivated with Agrobacterium for timesufficient to enable the transformation, for example 2 days. During thisstep, the Agrobacterium transfers the foreign gene construct intoCamelina sativa cells. The co-cultivated segments are then washed andplaced on Murashige and Skoog (MS) medium or an equivalent for callusand shoot regeneration.

[0065] The term “shoot and root regeneration” means the induction of theformation of shoots and roots from the transformed explants where shootsappear on the explants after growing the explants on culture mediumallowing shoot regeneration, preferably Murashige and Skoog (MS) mediumor an equivalent, supplemented with hormones and/or growth factorsallowing shoot and root regeneration, preferably cytokinin such as6-benzylaminopurine (BAP) and auxin such as α-naphthaleneacetic acid(NAA) and an effective amount of substance capable of preventing thegrowth of contaminants, such as antibiotics carbenicillin or morepreferably ticarcillin/clavulanic acid for time sufficient for shoots toappear.

[0066] The term “growing the shoots into a whole Camelina sativa plant”means that the regenerated transgenic shoots are grown and rooted forabout 2-3 weeks on a half strength Murashige and Skoog (MS) medium or anequivalent medium without hormones or optionally supplemented withauxins.

[0067] The term “hormones” and especially “plant hormones” or “growthfactors” mean organic compounds or molecules originating in certainparts or organs of a plant, which compounds when transported to anothertissue elicit a certain response. Plant hormones are active preferablyin small concentrations and can be used in different combinations. Themajor classes of plant hormones are auxin, gibberellins, cytokinins,ethylene, and abscisic acid, each of which has many effects. Also avariety of other compounds including oligosaccharins, batasins andbrassinosteroids function as hormones in plants.

[0068] “Hormones”, “plant hormones” and “growth factors” can be used assubstances or means in the transformation method to enchance thetransformation, selection, regeneration, growth or other functions.

[0069] Auxins can stimulate cellular elongation, differentiation ofvascular tissue, fruit development, formation of adventitious roots andproduction of ethylene. Naturally occuring and synthetic auxins includefor example indole-3-acetic acid (IAA), 4-chloro-IAA, phenylacetic acid,α-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D),indole-3-butyric acid (IBA), dimethylallylaminopurine (2iP) and otherauxins.

[0070] Gibberellins (GA for gibberellic acid) can stimulate extensivegrowth of intact plants, the transition from juvenile to adult growth,bolting of biennials, fruit formation, and germination of some cerealgrains. More than 80 gibberellins have been isolated from various fungiand plants including GA₃.

[0071] Cytokinins can stimulate cellular division, expansion ofcotyledons, and growth of lateral buds. Cytokinins also delay senescenceof detached leaves and, in combination with IAA, may influence formationof roots and shoots. Cytokinins include naturally occuring andartificial substances such as kinetin, zeatin, zeatin riboside,dihydrozeatin, isopentenyl adenine and 6-benzylaminopurine (BAP).

[0072] Ethylene is a gaseous hormone that can influence fruit ripening,abscission, sex expression, and the radial expansion of cells. Ethylenecan also function as a “wound hormone”. High amounts of ethylene areharmful, whereas low amounts promote rooting. Increased aeration of invitro cultures removes ethylene.

[0073] Abscisic acid (ABA) is an inhibitor that can cause stomata toclose, affects dormancy of some seeds, and, in general, counteracts thestimulatory effects of other hormones. These effects may occur becauseABA is calcium antagonist.

[0074] The term “a system for carrying out Agrobacterium mediatedgenetic transformation in Camelina sativa” means a system which in apackaged combination is intended for commercial use. Said systemcomprises Camelina sativa seeds, suitable DNA sequences and/or DNAconstructs, suitable media with optional additives and instructions forusing the transformation system. The Camelina sativa seeds can becultivated to provide the seedlings from which explants are taken. TheDNA sequence is a homologous or heterologous additional foreign gene orpart of a gene, as such, which encodes a desired product. The DNAsequences can also be provided as a DNA construct, in which case theforeign gene is functionally linked with one ore more optionalsequences, which are responsible for certain functions or capable ofregulating said functions. Examples of such sequences are promoters orsignal sequences. The DNA construct may comprise optional sequenceallowing selection of explants of Camelina sativa carrying thetransgenic inserts. The packaged combination can be provided with orwithout the media needed in the transformation procedure.

[0075] The term “assessing the efficacy of plant transformation” meansthe investigation of the rate of transgenic inclusions in transformedexplants and is assessed by recording by visible means including GUSexpression, PCR methods, Southern analysis or equivalent methods.

[0076] The term “homologous or heterologous recombinant products” meansproteins, peptides, metabolites, oils, carbohydrates, polymers, or otherproducts, which can be produced using Agrobacterium mediatedtransformation system in Camelina sativa. Homologous recombinant DNAproducts are produced when DNA sequences or genes native to Camelinasativa are used, whereas heterologous products are produced with DNAsequences or genes which are not naturally occuring in Camelina sativa.The “homologous and heterologous recombinant products” can originatefrom bacteria, viruses, fungi, plants and animals, including humanproteins and peptides which require processing.

GENERAL DESCRIPTION OF THE INVENTION

[0077] Brassica species have been used as common model plants in plantbreeding and molecular biology, but because they are prone to pests likeMeligethes aeneus, an alternative plant related to them would be useful.Camelina sativa would provide such a new model plant, which is notsensitive to the pest. Furthermore, Camelina sativa has a relativelysmall genome, including only 20 chromosomes, which simplifies its use ingenetic studies. Classically for example tobacco and Arabidopsis havebeen used as model plants and especially compared to Arabidopsis,Camelina sativa provides more plant material for further experiments.

[0078] In preliminary experiments we have examined different factors,which could have an effect on regeneration and transformation capacityof different explants of Camelina sativa. As a result we have developeda transformation method for plant explants, preferably leaf segments, ofCamelina sativa plants grown in vitro using Agrobacterium-mediatedtransformation.

[0079] The present invention thus provides an Agrobacterium-mediatedtransformation method of Camelina sativa.

[0080] The starting material for the transformation is Camelina sativaseed. Camelina sativa plants producing seed are grown in greenhouseconditions, since field grown plants produce seed contaminated withbacteria, which later prevents successful transformation withAgrobacterium. Camelina sativa seeds have a 0.5-1 mm thick hygroscopicpolysaccharide surface around the seed protecting the seed for exampleagainst fungal and bacterial spores and requiring more effective surfacesterilization compared to many other species. Seeds were firstoptionally sterilized and subsequently germinated and grown on Murashigeand Skoog (MS) agar medium or an equivalent plant growth medium.Preferably the leaf segments of 2-3 weeks, but preferably 10-20 days oldCamelina sativa plants preferably grown in vitro were used in theAgrobacterium-mediated transformation. Leaf segments were excised andthen placed on cultivation medium, preferebly Murashige and Skoog (MS)agar medium or an equivalent medium, supplemented with hormones, such ascytokinins and/or auxins or other hormones and sucrose or other sugarsource and cultivated on said medium for 24 hours.

[0081] The Camelina sativa explants were inoculated by immersingexplants in liquid Murashige and Skoog (MS) medium or an equivalentmedium or water containing Agrobacterium carrying the selectedtransformation vector with at least one gene foreign to said Camelinasativa.

[0082] After removing the redundant liquid on the immersed segments theexplants were placed on solid Murashige and Skoog (MS) medium or anequivalent supplemented with hormones, such as cytokinins or auxins, andoptionally with acetosyringone for co-cultivation. Explants wereco-cultivated with Agrobacterium for 2 days (48 hours) or a timesufficient to enable the transformation. During this step, theAgrobacterium transfered the foreign gene construct into Camelina sativacells. The co-cultivated segments were then washed and placed onMurashige and Skoog (MS) medium agar or equivalent medium for callus andshoot regeneration. Optional antibiotic selection begins preferablyimmediately after transformation and the result of the selection can beseen in 1-2 weeks, when explants begin to form callus. Selection wascarried out using optional antibiotics such as kanamycin, hygromycin orother selective agents for optionally 4-20 days or longer. An efficienttransformation was also obtained without selection with antibiotics.

[0083] When the selection had been completed the leaf segments had alsoproduced callus and shoots and roots. The regenerated, transgenic shootswere grown and rooted for about 2-3 weeks on Murashige and Skoog (MS)medium or equivalent medium, which is hormone-free or optionallysupplemented with auxins, including, but not limited to, indole-3-aceticacid (IAA), 4-chloro-IAA, phenylacetic acid, α-naphthaleneacetic acid(NAA) and/or 2,4-dichlorophenoxyacetic acid (2,4-D).

[0084] After rooting shoots were transferred to soil and transgenicplants were grown in greenhouse conditions (FIG. 5). Plants were testedfor GUS expression with a histological GUS assay and the presence of thetransgene was confirmed with PCR and Southern blot analysis.

[0085] The invention is also related to a system for carrying outAgrobacterium-mediated genetic transformation comprising a test kit in apackaged combination including one or more optionally sterilized seedsto provide seedlings from which explants of Camelina sativa areobtainable, an Agrobacterium vector, at least one DNA sequence encodinga desired gene product as such or in a recombinant DNA constructcomprising Agrobacterium and/or plasmids and at least one DNA sequenceencoding the desired gene product functionally combined with sequencesresponsible for or capable of regulating said functions, and optionallyat least one sequence allowing selection of explants of Camelina sativawith a culture of Agrobacterium carrying the said recombinant DNAconstruct, one or more cell culture mediums supplemented ornon-supplemented optionally with at least one hormone and/or growthfactor and/or at least one selective component which is capable ofselecting plant cells transformed with the said construct. The systemalso comprises the means and the method for obtaining whole transgenicCamelina sativa plants and growing them in vitro and in greenhouseincluding appropriate growth media, soil and equivalents.

[0086] The invention is described in more detail in the followingexperimental part. The scope of the invention is naturally notrestricted to these methods, one skilled in the art can easily replacethe suggested materials and methods with alternatives.

[0087] Materials and Methods

[0088] Plant material. The seeds of Camelina sativa were sterilized for1 min in 70% ethanol and 10 min in Na-hypochlorite (3% active Cl⁻) withaddition of Tween-20, and washed three times in sterile water. Thesterilized seeds were grown on Murashige and Skoog (MS; Murashige andSkoog, Physiol Plant. 15:472-493, 1962) agar medium or an equivalentmedium without sugars. The leaves of 2-3 weeks old, preferably 10-20days old, in vitro plants were used in the transformation experiments.

[0089] Agrobacterium vectors. Agrobacterium tumefaciens strain C58C1containing the plasmid pGV3850 (Zambryski et al., EMBO J. 2:2143-2150,1983), strain EHA105 (Hood et al., Transgenic Res. 2:208-218, 1993) withthe plasmid pTiBo542 and strain LBA4404 with pAL4404 (Hoekema et al.,Nature 303:179-180, 1983) were tested for transformation of Camelinasativa. The uidA marker gene (β-glucuronidase, GUS) containing an intron(uidA-int) (Vancanneyt et al., Mol. Gen. Genet. 220:245-250, 1990) wascloned into all vectors containing T-DNA region as listed above. TheuidA-intron-containing gene was used to prevent bacterial GUS expressionand enabled the testing of GUS-activity at an early stage oftransformation. The co-integrative pHTT294 vector, essentially similarto pHTT370 (Elomaa et al., Bio/Technology 11:508-511, 1993) carrying theuidA-intron-containing gene under the CaMV 35S promoter (Datla et al.,Plant Sci. 94:139-149, 1993), was transferred to an Agrobacterium strainC58C1. Binary pGPTV-HPT and pGPTV-KAN vectors (Becker et al., Plant.Mol. Biol. 20:1195-1197, 1992) with the uidA gene exchanged for theuidA-intron-containing gene under the control of the 35S promoter ofCaMV were transformed into Agrobacterium tumefaciens strains EHA105 andLBA4404.

[0090]Agrobacterium tumefaciens was grown overnight in liquid YEB(Lichtenstein and Draper, Genetic Engineering of Plants. In: Glover D M(ed.) DNA cloning—a practical approach, vol. 2. Oxford IRL, Oxford, pp67-119, 1985) medium with shaking supplemented with appropriateantibiotics for each strain. An aliquot ({fraction (1/100)} vol/vol) ofthe overnight culture was then inoculated in fresh YEB medium withappropriate antibiotics and bacteria were grown overnight with shaking.An Agrobacterium tumefaciens culture of OD₆₀₀=1.0 was used fortransformation.

[0091] Culture Medium

[0092] Composition of Murashige and Skoog (MS) plant growth medium:Salts: g/l Vitamins: mg/l NH₄NO₃ 1.65 Thiamine 0.1 KNO₃ 1.9 Pyridoxine0.1 MgS0₄x7H₂O 0.37 Nicotinic acid 0.5 KH₂P0₄ 0.17 Myo-inositol 100CaCl₂x2H₂O 0.44 Glycine 2.0

[0093] equivalent medium without sugars. The leaves of 2-3 weeks old,preferably 10-20 days old, in vitro plants were used in thetransformation experiments.

[0094] Agrobacterium vectors. Agrobacterium tumefaciens strain C58C1containing the plasmid pGV3850 (Zambryski et al., EMBO J. 2:2143-2150,1983), strain EHA105 (Hood et al., Transgenic Res. 2:208-218, 1993) withthe plasmid pTiBo542 and strain LBA4404 with pAL4404 (Hoekema et al.,Nature 303:179-180, 1983) were tested for transformation of Camelinasativa. The uidA marker gene (β-glucuronidase, GUS) containing an intron(uidA-int) (Vancanneyt et al., Mol. Gen. Genet. 220:245-250, 1990) wascloned into all vectors containing T-DNA region as listed above. TheuidA-intron-containing gene was used to prevent bacterial GUS expressionand enabled the testing of GUS-activity at an early stage oftransformation. The co-integrative pHTT294 vector, essentially similarto pHTT370 (Elomaa et al., Bio/Technology 11:508-511, 1993) carrying theuidA-intron-containing gene under the CaMV 35S promoter (Datla et al.,Plant Sci. 94:139-149, 1993), was transferred to an Agrobacterium strainC58C1. Binary pGPTV-HPT and pGPTV-KAN vectors (Becker et al., Plant.Mol. Biol. 20:1195-1197, 1992) with the uidA gene exchanged for theuidA-intron-containing gene under the control of the 35S promoter ofCaMV were transformed into Agrobacterium tumefaciens strains EHA105 andLBA4404.

[0095]Agrobacterium tumefaciens was grown overnight in liquid YEB(Lichtenstein and Draper, Genetic Engineering of Plants. In: Glover D M(ed.) DNA cloning—a practical approach, vol. 2. Oxford IRL, Oxford, pp67-119, 1985) medium with shaking supplemented with appropriateantibiotics for each strain. An aliquot ({fraction (1/100)} vol/vol) ofthe overnight culture was then inoculated in fresh YEB medium withappropriate antibiotics and bacteria were grown overnight with shaking.An Agrobacterium tumefaciens culture of OD₆₀₀=1.0 was used fortransformation.

[0096] Culture Medium

[0097] Composition of Murashige and Skoog (MS) plant growth medium:Salts: Vitamins: g/l mg/l NH₄N0₃ 1.65 Thiamine 0.1 KNO₃ 1.9 Pyridoxine0.1 MgS0₄x7H₂0 0.37 Nicotinic acid 0.5 KH₂P0₄ 0.17 Myo-inositol 100CaCl₂x2H₂0 mg/l g/l H₃B0₃ 6.2 Sucrose 2.0 MnSO₄x4H₂0 22.3 Agar 7.0ZnSOx7H₂0 8.6 KJ 0.83 pH 5.6 Na₂MoO₄x2H₂O 0.25 CuSO₄x5H₂O 0.025CoCl₂x2H₂0 0.025

[0098] Plant transformation. Leaf segments of in vitro grown Camelinasativa plants (FIG. 1) were cultivated for 24 hours on Murashige andSkoog (MS) medium or an equivalent medium supplemented with 0.7% agar.All MS culture media were supplemented with 2% sucrose and all in vitrocultures were kept at temperatures of 25° C. (day) and 18° C. (night)under 16 h photoperiod. Subsequently, the explants were immersed for 1-3min in Murashige and Skoog (MS) solution or an equivalent which had beeninoculated with a dilution (e.g. 1/10 vol/vol) of an overnight cultureof Agrobacterium tumefaciens. Thereafter, redundant liquid present onthe surface of leaf segments was removed using filter paper and theexplants were placed on the Murashige and Skoog (MS) agar mediumsupplemented with auxin and cytokinin hormones. 6-benzylaminopurine(BAP) and α-naphthaleneacetic acid (NAA), for co-cultivation withbacteria for 2 days. After co-cultivation, the explants were washed withwater containing cefotaxime (Claforan) (700 mg/l), carbenicillin (200mg/l) or ticarcillin/clavulanic acid (Duchefa) (100 mg/l). The surfacesof the explants were dried on filter paper and placed on the Murashigeand Skoog (MS) medium or an equivalent medium for selection and shootregeneration. The medium was supplemented with the same hormones andantibiotics than for transgenic tissue selection (kanamycin, hygromycin)and Agrobacterium growth inhibitors.

[0099] Selection and regeneration. Eventually, cultivation of theexplants for two weeks on Murashige and Skoog (MS) medium or anequivalent medium supplemented with 0.5-1.5 mg/l 6-benzylaminopurine(BAP) and 0.1-0.5 mg/l α-naphthaleneacetic acid (NAA) was found to bebest for callus, shoot and root formation. Thereafter, the wholeexplants or cut shoots were transferred to Petri dishes containinghormone-free culture medium, preferably Murashige and Skoog (MS) mediumor an equivalent medium, where recovered shoots elongated and started toroot. In the case that the explant forms both shoots and rootssimultaneously, whole explant is preferably transfered onto Murashigeand Skoog (MS) agar medium or an equivalent medium supplemented withcytokinins [1 mg/ml 6-benzylaminopurine (BAP)] to stimulate furthergrowth of shoots. Petri dishes were sealed with porous paper tape.Recovered transgenic shoots were grown on Murashige and Skoog (MS)medium or an equivalent medium without hormones or optionallysupplemented with 0.1-0.2 mg/l α-naphthaleneacetic acid (NAA) forstimulation of rooting, stem elongation and micropropagation. The exacthormone concentrations varied for different cultivars tested. Selectionusing hygromycin or alternatively kanamycin was applied preferablyimmediately after co-cultivation of the explants with Agrobacteriumtumefaciens. Antibiotics were used in concentrations ranging between15-25 mg/l. Selection with an antibiotic was carried out for 4-10 daysafter co-cultivation. It could be seen already after 5-7 days that theleaf segments produced callus and transgenic shoots.

[0100] Analysis of transgene expression. The histological GUS assay wasperformed on transformed callus and leaf tissue. TheuidA-intron-containing gene was used to prevent bacterial GUS expressionin transformation and to enable testing of GUS activity at early stagesof transformation, even immediately after co-cultivation withAgrobacterium tumefaciens. Usually in the optimization experiments, GUSassay was performed 4-7 days after co-cultivation with Agrobacteriumtumefaciens.

[0101] The transgenic plants which showed stable GUS expression and grewwell after selection were grown in the greenhouse (FIG. 5) They werethen used for PCR and Southern blot analysis to confirm thetransformation event at the DNA level.

[0102] Southern blot analysis was performed using a coding sequence ofuidA gene labeled with digoxigenin-11-UTP to obtain an RNA probeaccording to the manufacturer's instructions (Boehringer Mannheim).Three μg of DNA from Camelina sativa plants which showed stable GUSexpression was digested with EcoRI and BamHI restriction enzymes. Theseenzymes cut out a 2 kb uidA gene fragment from the T-region of pGPTV-HPTor pGPTV-KAN inserted in the Camelina sativa genome.

[0103] Results of the Preliminary Experiments in Developing theTransformation Method

[0104] Source plants. Field-grown Camelina sativa plants produce seedheavily contaminated and were practically improper for use in thetransformation, because leaf explants contained bacteria which preventedsuccessful transformation by Agrobacterium tumefaciens. To achieve goodstarting material seed producing Camelina sativa plants were grown ingreenhouse conditions. The seeds, which had been developed and maturedin greenhouse were free of contaminations after surface sterilization.Camelina sativa seeds have a hygroscopic polysaccharide surface, whichforms a 0.5-1 mm barrier around the seed. This barrier protects the seedagainst fungal and bacterial spores. This particular characteristics ofCamelina sativa seed surface requires more effective surfacesterilization of seeds compared to many other species. The sterilizationexperiments were performed as shown in Table 1. The Camelina sativaseeds were immersed in 70% ethanol for 1 min and treated withNa-hypochlorite solution with an addition of Tween-20 (1 drop per 100ml). TABLE 1 Seed germination (%) and contamination after differentsurface sterilization treatments. Concentration of Na-hypochlorite isshown in columns as % of active Cl. The time of treatment withNa-hypochlorite is shown in rows. Time min.\% Na-hyp. 1% Cl⁻ 3% Cl⁻ 6%Cl⁻  5 min. Contaminat. 100% 60% 10 min. 100% 100% 30% 20 min. 100%  60% 0%

[0105] After sterilization the seeds were washed three times insterilized water and placed on Murashige and Skoog (MS) agar medium oran equivalent medium without sugars for germination. Germination wasassessed 3 days after sterilization. The 10 min treatment with 3%Na-hypochlorite was found optimal for Camelina sativa seedsterilization.

[0106] Sterilized seeds were germinated and grown for 2-3 weeks orpreferably 10-20 days on Murashige and Skoog (MS) agar medium or anequivalent medium without sucrose and hormones. The green leaves servedas a source for explants for the transformation.

[0107] Plant transformation. Three different Agrobacterium tumefaciensstrains, namely C58C1, EHA105 and LBA4404 were tested. C58C1pGV3850harbors the cointegrative vector pHTT294. The strains EHA105 and LBA4404carried the binary vector pGPTV-HPT. UidA-intron-containing reportergene was cloned from pGUS-int into all the binary and cointegrativevectors used in the transformation experiments. The uidA-int gene wasplaced under CaMV 35S promoter.

[0108] Hypocotyl, cotyledon, leaf and stem segments were tested foraffinity to Agrobacterium tumefaciens. The cotyledon and leaf segmentshad the best transformation capacity. Because of their betterregeneration ability, leaf segments were used in further transformationexperiments. Leaves of in vitro grown Camelina sativa plants are rathersmall in size: 2 to 4 cm long and 0.5-1 cm wide. Therefore, narrowleaves were cut only across the leaf while larger leaves were also cutin half along the central vein.

[0109] Transformation efficiencies of different Agrobacteriumtumefaciens strains were measured as a proportion of blue inclusions incallus one week after inoculation of leaf segments (FIGS. 3a, 3 b).TABLE 2 Transformation efficiencies of different Agrobacteriumtumefaciens strains. First column: GUS positives/all explants, Secondcolumn: intensive transformation. Blue inclusions IntensivelyTransformation Agrobacterium all explants transformed % (intensive)LBA4404pGPTV-HPT 35/50 7/50 70% (14) EHA105pGPTV-HPT 24/50 48% (0)C58C1pGV3850 pHTT294 33/50 4/50 66% (8)

[0110] The results of the three transformation experiments, summarizedin Table 2, showed that LBA4404 and C58C1pGV3850 strains were effectivein transforming Camelina sativa. EHA105 was slightly less effective. Theexplants infected with LBA4404 or C58C1 strains had large intensivelystained blue inclusions. Thus, the strains LBA4404 and C58C1 were usedin subsequent transformation experiments.

[0111] Shoot regeneration. Effects of different hormones on variousexplants of Camelina sativa (hypocotyl, cotyledon, leaf and stemsegments) were tested in preliminary experiments to achieve sufficientshoot regeneration. 6-benzylaminopurine (BAP) and α-naphthaleneaceticacid (NAA) were more effective to induce shoot and root regenerationthan kinetin and indole-3-acetic acid (IAA). The regeneration capacityof cotyledons was 30-50% whereas shoots from hypocotyl and stem segmentsdid not regenerate. The best regeneration (100%) was achieved with leafsegments (FIG. 2). The 2,4-dichlorophenoxyacetic acid (2,4-D),gibberellins as well as silver nitrate treatments did not have an effecton shoot regeneration. The best regeneration was achieved with a certainratio of auxin and cytokinin hormones. For example, the best shootregeneration of leaf segments of Camelina sativa variety cv. Calena wasachieved with the hormone combination of 1 mg/l 6-benzylaminopurine(BAP) and 0.2 mg/l α-naphthaleneacetic acid (NAA), while the optimalcombination for Camelina sativa variety cv. Calinca was 0.7 mg/l6-benzylaminopurine (BAP) and 0.3 mg/l α-naphthaleneacetic acid (NAA).

[0112] Recovered shoots had a tendency for inflorescence formation andhad problems with rooting. To overcome these problems, recovered shootswere cultivated subsequently on Murashige and Skoog (MS) medium or anequivalent medium optionally supplemented with auxins (e.g.indole-3-acetic acid (IAA) 1 mg/l). Another way was to regenerate shootsand roots simultaneously with the hormone combination of 0.5-1 mg/l6-benzylaminopurine (BAP) and 0.2-0.7 mg/l α-naphthaleneacetic acid(NAA).

[0113] Several different factors were tested for impact on shootregeneration efficiency. Optimal parameters were found for pH (5.6-5.8),for sucrose content (2-3%), and solidifiers (0.7% agar). Modificationsin the concentration of NH₄, NO₃ ⁻, K⁺ and Ca²⁺ ions in the standardMurashige and Skoog (MS) medium had no effect nor did the addition ofglucose. Culturing the explants on the B5 medium had also no effect onshoot regeneration.

[0114] Selection. To prevent Agrobacterium tumefaciens growth on themedium, cefotaxime (Claforan) (500 mg/l), carbenicillin (200 mg/l),ticarcillin/clavulonic acid (Duchefa) (100 mg/ml) or vancomycin (200mg/ml) were used.

[0115] The selection markers i.e. the hpt and nptII genes intransformation constructs provided the plants with resistance tohygromycin and kanamycin, respectively. It had been found that theapplication of a selection pressure (15-20 mg/l, preferably 10-20 mg/lof antibiotic) preferably for 4-10 days after washing of theAgrobacterium tumefaciens from explants is optimal. First regenerativeprimordia form on the calli already 10 days after cutting of the leafsegments, and selection of transformed tissues should be performedbefore that. It was found in preliminary experiments that 5-15 mg/lantibiotic prevented morphogenesis of explants. Selection of transformedtissue using 5-10 mg/l hygromycin or kanamycin was not enough. On theother hand, the concentrations of the antibiotic higher than 20-30 mg/lkilled the explants too fast for any shoots to recover.

[0116] Analysis of Transformation

[0117] The histological GUS assay was performed on transformed callusand leaf tissue. To prevent GUS expression in Agrobacterium tumefaciens,the uidA gene containing the intron was used in transformationexperiments. It enabled the testing of GUS activity almost immediatelyafter co-cultivation with Agrobacterium tumefaciens. Usually, GUS assaywas made 4-7 days after co-cultivation with Agrobacterium tumefaciensduring the optimization of transformation (FIG. 3). The assay was alsoperformed on regenerated primordia and shoots as well as leaf segmentsof recovered plants.

EXAMPLE 1

[0118] Transformation Protocol for Camelina sativa cv. Calena withAgrobacterium tumefaciens strain LBA4404 Harboring the Binary PlasmidpGPTV-HPT with uidA Intron Containing Gene.

[0119] The seeds of Camelina sativa plant grown in greenhouse weresterilized by immersing in 70% ethanol for 1 min and then treating for10 min with Na-hypochlorite solution (3% active Cl⁻) with an addition ofTween-20 (1 drop per 100 ml). After sterilization the seeds were washedthree times in sterile water and placed on solid Murashige and Skoog(MS) agar medium (Murashige and Skoog, Physiol. Plant. 15:472-493, 1962)without sugars for germination. Sterilized seeds were germinated andgrown 2-3 weeks on solid Murashige and Skoog (MS) medium withouthormones (FIG. 1). Green leaves served as a source of explants fortransformation procedure.

[0120]Agrobacterium tumefaciens strain LBA4404 carrying pGPTV-HPT-GUSintvector was grown overnight at 28° C. with shaking in liquid YEB medium(Lichtenstein and Draper, Gene Engineering of Plants. In: Glover D M(ed.) DNA Cloning—A Practical Approach, vol. 2. Oxford IRL, Oxford, pp67-119, 1985) supplemented with 50 mg/l kanamycin and rifampicin.Subsequently an aliquot of the culture ({fraction (1/100)} v/v) wasinoculated in fresh YEB medium supplemented with 50 mg/l kanamycin andrifampicin and the bacteria were grown overnight with shaking.Agrobacterium culture of OD₆₀₀=1.0 was used in the transformationexperiments.

[0121] Narrow leaves of in vitro grown Camelina sativa plants were cutonly across the leaf blade, whereas large leaves were additionally cutin half along the central vein. The leaf segments were cultivated for 24hours on Murashige and Skoog (MS) 0.7% agar medium supplemented with 1mg/l 6-benzylaminopurine (BAP) and 0.2 mg/l α-naphthaleneacetic acid(NAA). All the Murashige and Skoog (MS) culture media were supplementedwith 2% sucrose and all in vitro cultures were kept at temperatures of25° C. (day) and 18° C. (night) under the photoperiod of 16 h. Theexplants were immersed for 1-3 min in Murashige and Skoog (MS) solutioninoculated with a dilution (e.g. 1/10 v/v) of the overnight culture ofAgrobacterium tumefaciens LBA4404. Redundant liquid on the stem segmentswas removed using filter paper and the explants were placed on Murashigeand Skoog (MS) agar medium supplemented with auxin and cytokinin forco-cultivation with bacteria for 2 days. The explants were washed withwater containing claforan [cefotaxime) (700 mg/l)] or carbenicillin (700mg/ml). The surfaces of the explants were dried on filter paper and theexplants were placed on Murashige and Skoog (MS) medium supplementedwith hormones [0.7 mg/l 6-benzylaminopurine (BAP), 0.25 mg/lα-naphthaleneacetic acid (NAA)] and 200 mg/l carbenicillin or claforanand 15 mg/ml hygromycin. Two to three weeks old shoots (FIG. 2) wereplaced to the normal or half strength Murasghige and Skoog (MS) mediumsolidified with 0.7% agar and supplemented with 200 mg/l carbenicillinor cefotaxime and optionally with 15 mg/l hygromycin and auxin[indole-3-acetic acid (IAA) 0.5-1 mg/l] shoots were transferred to soiland transgenic plants were grown in greenhouse conditions (FIG. 5).Transgenic plants were tested for uidA (GUS) gene expression with ahistological GUS assay and the presence of the transgene was confirmedwith Southern analysis.

EXAMPLE 2

[0122] Transformation Protocol for Camelina sativa cv. Calinca withAgrobacterium tumefaciens strain C58C1 pGV3850 Harboring the Binary TiVector with Kanamycin Selection.

[0123] Seeds were taken from greenhouse grown Camelina sativa cv.Calinca plants (no older than 4 months). Transformation efficiencyincreases from 66% to 100% if donor plants are grown in greenhouse.

[0124] 10 Days Before Excision of the Explants.

[0125] Seeds of Camelina sativa were sterilized and placed in vitro onMurashige and Skoog (MS) agar medium without sucrose and grown attemperatures of 25° C. (day) and 18° C. (night) as described in Example1.

[0126] 1^(st) Day.

[0127] A fresh colony of Agrobacterium tumefaciens strain C58C1pGV3850carrying binary pGPTV-KAN vector (Becker et al., Plant Mol. Biol.20:1195-1197, 1992) containing uidA-int gene under 35S promoter andselectable marker gene nptII, was inoculated in 3 ml of liquid YEBmedium supplemented with 25 mg/l rifampicin (Rif) and 50 mg/l kanamycin(Kan). The bacteria were grown overnight with shaking at 28° C.

[0128] 2^(d) Day. Pre-Cultivation.

[0129] The first leaves (no cotyledons) of in vitro grown Camelinasativa were cut into segments across the leaf and were placed onpre-cultivation plates containing 0.7% MS agar medium supplemented with2% sucrose, 0.7 mg/l 6-benzylaminopurine (BAP) and 0.3 mg/lα-naphthaleneacetic acid (NAA). All dishes were sealed with porous papertape (Micropore 3M).

[0130] A 30 μl aliquot of overnight culture of the Agrobacteriumtumefaciens was inoculated in 3 ml of fresh YEB medium supplemented withrifampicin (Rif) and kanamycin (Kan).

[0131] 3^(d) Day. Agrobacterium tumefaciens Inoculation.

[0132] The explants were immersed in liquid Murashige and Skoog (MS)medium supplemented with 2% sucrose and inoculated with a {fraction(1/10)} (v/v) dilution of the overnight culture of Agrobacteriumtumefaciens. After 5 min inoculation redundant liquid on the explantswas removed using sterilized filter paper.

[0133] The explants were placed on Murashige and Skoog (MS) mediumsupplemented with 2% sucrose for co-cultivation with the Agrobacteriumtumefaciens for two days at 28° C. in dim light.

[0134] 5^(th) Day. Washing and Selection.

[0135] The explants were washed with water containing 100 mg/lticarcillin/clavulanic acid (Duchefa). Ticarcillin (Tc) has lessnegative effect on shoot and root regeneration than cefotaxime(Claforan) and carbenicillin. On the other hand it is more effectivegrowth inhibitor of Agrobacterium tumefaciens than vancomycin. Theexplants were dried on the filter paper and transferred onto selectionmedium containing 0.7% Murashige and Skoog (MS) agar medium supplementedwith 2% sucrose, 0.7 mg/l 6-benzylaminopurine (BAP), 0.3 mg/lα-naphthaleneacetic acid (NAA), 15 mg/l kanamycin and 50 mg/lticarcillin/clavulanic acid (Duchefa). The explants were cultured on theselection medium for 4-5 days.

[0136] 10^(th) Day. Regeneration.

[0137] The explants were transferred onto plates containing 0.7% MS agarmedium supplemented with 2% sucrose, 0.7 mg/l 6-benzylaminopurine (BAP),0.3 mg/l α-naphthaleneacetic acid (NAA), and 50 mg/lticarcillin/clavulanic acid (Duchefa) for shoot and root regenerationfor 10-14 days. Tall (3 cm high) plates were sealed with porous papertape to increase aeration. Simultaneous regeneration of shoots and rootsis preferable for effective recovery of transgenic Camelina sativaplants.

[0138] 20-24^(th) Day. Shoot and Root Elongation.

[0139] Explants forming 0.5-1 cm long leaves (shoots) and roots weretransferred on 0.7% Murashige and Skoog (MS) agar medium containing 2-3%sucrose and 100 mg/l ticarcillin/clavulanic acid without hormones oroptionally supplemented with 1 mg/ml 6-benzylaminopurine (BAP) for 5-7days. Alternatively explants were transferred to fog system (mistchamber) in greenhouse for consecutive growth.

[0140] 25-30^(th) Day. Transgenic Plant Growth.

[0141] Successfully grown and rooted shoots were transferred to soilwithout separation from explants. Shoots in pots were placed into closedchamber. The chamber was opened gradually day by day to increaseaeration. Alternatively explants were transferred to fog system (mistchamber) in greenhouse for consecutive growth. The recovered shootsformed inflorescence and seedpods. Plant tissues were tested forexpression of marker gene (GUS) with GUS assay, PCR and Southern blot.

EXAMPLE 3

[0142] Transformation Protocol for Camelina sativa cv. Calena withAgrobacterium tumefaciens Strain C58C1 pGV3850 Harboring CointegrativeTi DNA Without Selection of Transgenic Tissues.

[0143] Seeds were taken from greenhouse grown Camelina sativa cv. Calenaplants (no older than 4 months). Transformation efficiency increasesfrom 66% to 100% if donor plants are grown in greenhouse.

[0144] 10 Days Before Explants Excision.

[0145] Seeds were sterilized and placed in vitro on Murashige and Skoog(MS) medium without sucrose and grown at temperatures of 25° C. (day)and 18° C. (night) as described in Example 1.

[0146] 1^(st) Day.

[0147] A fresh colony of C58C1pGV3850 with interned Ti DNA from pHTT-HPT(Elomaa et al., Bio/Technology 11:508-511, 1993) vector containing GUSgene under 35S promoter and hpt selectable marker was inoculated in 3 mlof liquid YEB supplemented with 25 mg/l rifampicin (Rif) and 100 mg/lspectinomycin (Spe) or streptomycin (Str). The bacteria were grownovernight with shaking at 28° C.

[0148] 2^(nd) Day. Pre-Cultivation.

[0149] The first leaves (no cotyledons) were cut into segments acrossthe leaf and placed onto the pre-cultivation plates containing 0.7%Murashige and Skoog (MS) agar medium with 2% sucrose supplemented with 1mg/l 6-benzylaminopurine (BAP) and 0.5 mg/l α-naphthaleneacetic acid(NAA). All plates were sealed with porous paper tape (Micropore 3M).

[0150] A 30 μl aliquot of overnight culture of the Agrobacteriumtumefaciens was inoculated in 3 ml of fresh YEB medium supplemented withrifampicin (Rif), spectinomycin (Spe) or streptomycin (Str).

[0151] 3^(rd) Day. Agrobacterium Inoculation.

[0152] The plant explants were immersed in liquid Murashige and Skoog(MS) medium supplemented with 2% sucrose and inoculated with a {fraction(1/10)} dilution of the overnight culture of Agrobacterium tumefaciens.Redundant liquid on the explants was removed on sterilized filter paper.The explants were co-cultivated with the Agrobacterium tumefaciens fortwo days at 28° C. in dim light.

[0153] 5^(th) Day. Washing and Reegneration.

[0154] The explants were washed with water containing 100 mg/lticarcillin/clavulanic acid (Duchefa). Ticarcillin (Tc) has lessnegative effect on shoot and root regeneration compared to cefotaxime(Claforan) and carbenicillin. On the other hand it is more effectivegrowth inhibitor of Agrobacterium tumefaciens than vancomycin. Theexplants were dried on the filter paper. Then the explants were placedonto selection medium plates containing 7% Murashige and Skoog (MS) agarmedium with 2% sucrose supplemented with 1 mg/l 6-benzylaminopurine(BAP), 0.5 mg/l α-naphthaleneacetic acid (NAA) and 50 mg/lticarcillin/clavulanic acid (Duchefa) 0.5 mg/l for shoot and rootregeneration for 2-3 weeks. Tall (3 cm high) plates were sealed withporous paper tape to increase aeration.

[0155] 20-24^(th) Day. Shoot and Root Elongation.

[0156] Explants forming 0.5-1 cm long leaves (shoots) and roots weretransferred onto 0.7% Murashige and Skoog (MS) agar medium containing 2%sucrose supplemented with 100 mg/l ticarcillin/clavulanic acid (Duchefa)without hormones or with 1 mg/ml 6-benzylaminopurine (BAP) for 5-7 days.Plates were not sealed with tape.

[0157] Regenerated shoots were tested for GUS expression withhistological GUS assay. GUS activity was seen in 4 shoots out of 123. Itmeans that average of about 3% of shoots regenerated aftertransformation were transgenic without the use of antibiotic selection.Thus the method can be used for producing transgenic Camelina sativaplants free from antibiotic resistance genes or selectable marker genes.

[0158] The strain C58C1pGV3850 was the most effective for transformationof Camelina sativa. 100% of the explants were transformed. The averageproportion of tissue in each explant showing GUS expression was morethan 30%. This is the highest level of transformation that wasregistered by present inventors. The transformation efficiency enablesto obtain transgenic plats without antibiotic or other selection oftransgenic plants.

EXAMPLE 4

[0159] Analysis of Transformation.

[0160] The histological GUS assay was performed on transformed callusand leaf tissue. To prevent GUS expression in Agrobacteria the uidA genecontaining an intron was used in transformation experiments. It enabledthe testing of GUS activity even immediately after co-cultivation withAgrobacterium tumefaciens. Usually, GUS assay was made 4-7 days afterco-cultivation with Agrobacterium tumefaciens during the optimization oftransformation (FIG. 3). The assay was also performed on regeneratedprimordia and shoots as well as leaf segments of recovered plants.

[0161] Transgenic plants which showed steady positive GUS expression andgrew well under selection conditions were used for PCR analysis oftransgene insertion and Southern blot analysis to confirm thetransformation events.

[0162] PCR Analysis.

[0163] Total genomic DNA was isolated from leaf tissue of transgenic andnon-transgenic Camelina sativa plants using DNeasy Plant Mini Kitaccording to the supplier's instructions (Qiagen). The presence of theuidA and hpt gene in the GUS positive plants was determined by PCRanalysis using 24 nucleotides long primers specific to the codingsequences of uidA and hpt genes. PCR reaction mix containedapproximately 1 ng/μl of template DNA and DyNAzyme polymerase(Finnzymes) was used for amplification. PCR program conisted of: 94° for2 min; 30 cycles of 94° C. for 30 sec, 48° C. for 30 sec and 72° C. for2 min. Three μl of PCR reaction mixture was run in 0.8% agarose gelcontainig ethidium bromide at 100 V. No PCR product was obtained whennon-transgenic Camelina sativa DNA was used as template, whereas whenusing transgenic Camelina sativa an amplification product of 700nucleotides corresponding to the positive control was obtained whichconfirmed the presence of transgene in transgenic Camelina sativa plants(FIG. 4).

[0164] Southern Analysis

[0165] Total genomic DNA was isolated from leaf tissue of Camelinasativa plants using DNeasy Plant Midi Kit according to the supplier'sinstructions (Qiagen). Three μg of DNA from GUS positive Camelina sativaplants was digested with EcoRI and BamHI restriction enzymes. Theseenzymes cut out a 2 kb uidA gene fragment from the T-region of pGPTV-KAN(-HPT) inserted in the plant genome. Digested DNA samples were separatedin a 0.7% agarose (Promega) gel overnight at 15 mA current andtransferred to positively charged nylon membrane (Boehringer Mannheim)using vacuum blotter. RNA probes were synthesized using T3 RNApolymerase on the pBluescript vector carrying uidA or hpt gene sequenceand labeled with digoxigenin-11-UTP. The membrane was hybridized anddeveloped according to the supplier's instructions (Boehringer Mannheim,The DIG user's guide for filter hybridization). The membrane wasprehybridized at 50° C. for 2 h and hybridized at 50° C. in a “DIG EasyHyb” hybridization solution (Boehringer Mannheim) overnight with adigoxigenin-UTP labeled RNA probe. The concentration of RNA probe was100 ng/ml. After hybridization the membrane was washed in SSC buffers,blocked and detected using “Anti-Digoxigenin-AP alkaline phosphatase(Boehringer Mannheim). Chemiluminescent detection was done with withCSPD-substrate and the membrane was exposed to X-ray film (BoehringerMannheim). Presence of the transgene insertion was proved in comparisonto DNA of non-transgenic Camelina sativa plant DNA as negative control,and to plasmid DNA carrying the gene sequence mixed with non-transgenicplant DNA as positive control.

A SHORT DESCRIPTION OF THE DRAWINGS

[0166]FIG. 1 shows in vitro cultured Camelina sativa plant.

[0167]FIG. 2 shows regenerated shoots of Camelina sativa on leaf segmentexplants.

[0168]FIG. 3a depicts GUS expression in callus tissue of Camelinasativa. The arrowheads point to GUS stained inclusions.

[0169]FIG. 3b depicts GUS expression in callus tissue of Camelinasativa. The arrowheads point to GUS stained inclusions.

[0170]FIG. 4 shows results of PCR amplification of a transgenicinsertion. The PCR was carried out using specific primers developed forthe central part of uidA gene. The length of the DNA sequence betweenthe primers is about 700 nucleotides and thus the size of theamplification product is also 700 nucleotides. Samples on the gel aremarked as follows: NT, non-transgenic Camelina sativa (negativecontrol); T, transgenic GUS positive line of Camelina sativa expressinguidA gene; +, uidA gene sequence cloned in pBluescript vector aspositive control. M is a one kilobase (1 kb) marker ladder (Fermentas).The sizes of some of the marker bands are shown on the left side of thefigure. No PCR product was obtained when non-transgenic Camelina sativaDNA was used as template, whereas when using transgenic Camelina sativaan amplification product of 700 nucleotides corresponding to thepositive control was obtained.

[0171]FIG. 5 shows Camelina sativa plantlets grown in greenhouseconditions. The plantlets are transgenic shoots recovered and rootedafter in vitro selection of transformed explants of Camelina sativa.

DETAILED DESCRIPTION OF THE INVENTION

[0172] In the present invention the terms used have the meaning theygenerally have in the fields of conventional plant breeding, plantbiochemistry and production of transgenic plants, including recombinantDNA technology as well as agriculture and horticulture. Some terms,however, are used with a somewhat deviating or broader meaning in thiscontext. Accordingly, in order to avoid uncertainty caused by terms withunclear meaning some of the terms used in this specification and in theclaims are defined in more detail below.

[0173] Abbreviations

[0174] BAP 6-benzylaminopurine

[0175] 2,4-D 2,4-dichlorophenoxyacetic acid

[0176] GUS β-glucuronidase (uidA reporter gene)

[0177] Hpt gene encoding for hygromycin phosphotransferase

[0178] Hyg hygromycin

[0179] IAA indole-3-acetic acid

[0180] Kan kanamycin

[0181] MS Murashige and Skoog medium

[0182] NAA α-naphthaleneacetic acid

[0183] nptII gene encoding for neomycin phosphotransferase II

[0184] Rif rifampicin

[0185] Spe spectinomycin

[0186] Str streptomycin

[0187] Tc ticarcillin

[0188] uidA gene encoding for β-glucuronidase (GUS)

[0189] YEB medium for cultivation of Agrobacterium cells

[0190] Definitions

[0191] “Camelina sativa” belongs to the family of Brassicaceae in thetribe Sisymbriae. The seed yields of Camelina sativa are comparable tothe seed yields of oil seed rape. Useful varieties of Camelina sativaare for example var. Calina and var. Calinca which are hereby mentionedbut not claimed.

[0192] The term “Agrobacterium” means Agrobacterium tumefaciens,Agrobacterium rhizogenes or another Agrobacterium species useful forgenetic transformation of plants to produce genetically modified plants.

[0193] “Agrobacterium tumefaciens” is a naturally occuring bacteriumwhich when containing a circular Ti (Tumor inducing) plasmid is able toform crown gall disease in many species of dicotyledonous plants. Crowngall occurs when a wound is invaded by Agrobacterium. Agrobacteriumtumefaciens natively has the ability to transfer a portion of its DNAcalled T-DNA, into the genome of the plant cells. InAgrobacterium-mediated transformation T-DNA is replaced with a foreignset of genes, thus, making the bacterium capable of transferring theforeign genes into the genome of the plant cell. “Agrobacteriumtumefaciens” can be one of the three different strains of Agrobacteriumused in the transformation of Camelina sativa or an equivalent strainsuitable for the transformation. The strain LB4404 carries the plasmidpAL4404, the strain C58C1 carries the plasmid pGV3850 and the strainEHA105 carries the plasmid pTiBo542.

[0194] The term “Agrobacterium-mediated genetic transformation” in thepresent invention means that Agrobacterium is used as a vector which isable to transfer foreign gene(s) to Camelina sativa cells. The T-DNAportion of the Ti plasmid is replaced with the foreign gene and, afterthe Agrobacterium infection, transferred into the plant chromosomal DNA.

[0195] The term “transgenic plant” means a plant, especially Camelinasativa plant, which is obtained using the method disclosed in thepresent invention. The optionally sterilized seeds of Camelina sativaare germinated and grown to plants, which provide explants for use inAgrobacterium-mediated transformation method. An Agrobacterium straincontaining an Agrobacterium vector comprising at least one recombinantDNA construct and an optional selectable marker gene is allowed totransform Camelina sativa cells in a cell culture medium optionallysupplemented with at least one hormone and/or growth factor. Selectionon a medium optionally containing at least one selective component isfollowed by the regeneration of one or more shoots or roots from thetransformed explants on a cell culture medium optionally containing atleast one hormone and/or growth factor. The shoots are grown into wholetransgenic Camelina sativa plants.

[0196] The term “explant” means a part or a piece which is taken from aplant, in this this context from Camelina sativa. These pieces or tissueexplants can be excised from hypocotyl, cotyledon, stem, leaf or otherplant organs and can be used for in vitro culture and for transformationexperiments. The term “in vitro explant” means a Camelina sativa explantexcised from hypocotyl, cotyledon, stem, leaf or other plant organsoriginating from plants grown in vitro preferably under sterileconditions on culture media. An “explant” or “in vitro explant” can be aleaf segment.

[0197] The term “leaf segment” means a piece of leaf from preferably invitro grown Camelina sativa. The leaves of in vitro grown Camelinasativa can be rather small in size for example 2-4 cm long and 0.5-1 cmwide. Accordingly, narrow leaves are cut across the leaf while largerleaves are also cut in half along the central vein.

[0198] The term “recombinant DNA construct” means a DNA sequenceincluding linear or circular vector, plasmid or insert created byligating or joining together pieces of DNA that are not normallycontiguous in nature. The construct, which is transfered into the plantcell, comprises a specific gene of interest, which is desired to beintroduced into the germline of the plant, and an optional selectablemarker gene that confers upon the plant cell a resistance to a chemicalselective component (selection agent).

[0199] The term “selectable marker gene” means an optionally used genein plant transformation such as the gene for neomycin phosphotransferase(npt II), which expresses an enzyme conferring resistance to theantibiotic kanamycin and the related antibiotics neomycin, paromomycin,gentamicin, and G418, or the gene for hygromycin phosphotransferase(hpt), which expresses an enzyme conferring resistance to hygromycin.Other selectable marker genes include genes encoding herbicideresistance, metal resistance or sensitivity, for example Cu-resistanceor sensitivity, genes utilizing special carbohydrate sources or othermetabolites including for example mannose or other selection systems.

[0200] The term “selection of the transformed tissue of Camelina sativa”means that the transformed tissue is grown on a medium containing asubstrate allowing the selection of transformed tissues which carry amarker gene. Preferred selective substances are antibiotics, for examplehygromycin or kanamycin, but other selection systems such as herbicides,metal resistance or sensitivity, including e.g. Cu-resistance orsensitivity and special carbohydrate sources or other metabolites areapplicable. The use of a marker gene e.g. hpt or nptII is optional,since possible harmful effects of the marker genes used with plantcloning vectors is one area of concern with genetically engineeredplants. Antibiotic selection begins preferably immediately aftertransformation and the result of the selection can be seen in 1-2 weeks,when the explants begin to form callus.

[0201] The term “Murashige and Skoog (MS) medium or equivalent” or “MSmedium or equivalent” means that preferably Murashige and Skoog's growthmedium, that is MS medium, is used in the method of the presentinvention (Murashige and Skoog, Physiol Plant. 15:472-493, 1962). Anyother medium suitable for the purposes of the present invention can alsobe used including for example the B5 medium. The growth medium can be inliquid form or made solid or semisolid using an appropriate amount ofagar or gelrite for example 0.7 g/l. The concentration of the medium canoptionally varies from half strength to 2× strength.

[0202] The term “inoculation with Agrobacterium” means that the Camelinasativa explants are inoculated with bacteria by placing them inAgrobacterium suspension to enable the transformation of Camelina sativawith Agrobacterium. Before inoculation an overnight culture ofAgrobacterium has been diluted in a Murashige and Skoog (MS) solution oranother suitable growth medium to enable the transformation of Camelinasativa with Agrobacterium.

[0203] The term “co-cultivation” means that Camelina sativa explants areplaced preferably on solid Murashige and Skoog (MS) agar medium or anequivalent medium supplemented with at least one hormone, such ascytokinin or auxin, and optionally with acetosyringone forco-cultivation. Explants are co-cultivated with Agrobacterium for timesufficient to enable the transformation, for example 2 days. During thisstep, the Agrobacterium transfers the foreign gene construct intoCamelina sativa cells. The co-cultivated segments are then washed andplaced on Murashige and Skoog (MS) medium or an equivalent for callusand shoot regeneration.

[0204] The term “shoot and root regeneration” means the induction of theformation of shoots and roots from the transformed explants where shootsappear on the explants after growing the explants on culture mediumallowing shoot regeneration, preferably Murashige and Skoog (MS) mediumor an equivalent, supplemented with hormones and/or growth factorsallowing shoot and root regeneration, preferably cytokinin such as6-benzylaminopurine (BAP) and auxin such as α-naphthaleneacetic acid(NAA) and an effective amount of substance capable of preventing thegrowth of contaminants, such as antibiotics carbenicillin or morepreferably ticarcillin/clavulanic acid for time sufficient for shoots toappear.

[0205] The term “growing the shoots into a whole Camelina sativa plant”means that the regenerated transgenic shoots are grown and rooted forabout 2-3 weeks on a half strength Murashige and Skoog (MS) medium or anequivalent medium without hormones or optionally supplemented withauxins.

[0206] The term “hormones” and especially “plant hormones” or “growthfactors” mean organic compounds or molecules originating in certainparts or organs of a plant, which compounds when transported to anothertissue elicit a certain response. Plant hormones are active preferablyin small concentrations and can be used in different combinations. Themajor classes of plant hormones are auxin, gibberellins, cytokinins,ethylene, and abscisic acid, each of which has many effects. Also avariety of other compounds including oligosaccharins, batasins andbrassinosteroids function as hormones in plants. “Hormones”, “planthormones” and “growth factors” can be used as substances or means in thetransformation method to enchance the transformation, selection,regeneration, growth or other functions.

[0207] Auxins can stimulate cellular elongation, differentiation ofvascular tissue, fruit development, formation of adventitious roots andproduction of ethylene. Naturally occuring and synthetic auxins includefor example indole-3-acetic acid (IAA), 4-chloro-IAA, phenylacetic acid,α-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D),indole-3-butyric acid (IBA), dimethylallylaminopurine (2iP) and otherauxins.

[0208] Gibberellins (GA for gibberellic acid) can stimulate extensivegrowth of intact plants, the transition from juvenile to adult growth,bolting of biennials, fruit formation, and germination of some cerealgrains. More than 80 gibberellins have been isolated from various fungiand plants including GA₃.

[0209] Cytokinins can stimulate cellular division, expansion ofcotyledons, and growth of lateral buds. Cytokinins also delay senescenceof detached leaves and, in combination with IAA, may influence formationof roots and shoots. Cytokinins include naturally occuring andartificial substances such as kinetin, zeatin, zeatin riboside,dihydrozeatin, isopentenyl adenine and 6-benzylaminopurine (BAP).

[0210] Ethylene is a gaseous hormone that can influence fruit ripening,abscission, sex expression, and the radial expansion of cells. Ethylenecan also function as a “wound hormone”. High amounts of ethylene areharmful, whereas low amounts promote rooting. Increased aeration of invitro cultures removes ethylene.

[0211] Abscisic acid (ABA) is an inhibitor that can cause stomata toclose, affects dormancy of some seeds, and, in general, counteracts thestimulatory effects of other hormones. These effects may occur becauseABA is calcium antagonist.

[0212] The term “a system for carrying out Agrobacterium mediatedgenetic transformation in Camelina sativa” means a system which in apackaged combination is intended for commercial use. Said systemcomprises Camelina sativa seeds, suitable DNA sequences and/or DNAconstructs, suitable media with optional additives and instructions forusing the transformation system. The Camelina sativa seeds can becultivated to provide the seedlings from which explants are taken. TheDNA sequence is a homologous or heterologous additional foreign gene orpart of a gene, as such, which encodes a desired product. The DNAsequences can also be provided as a DNA construct, in which case theforeign gene is functionally linked with one ore more optionalsequences, which are responsible for certain functions or capable ofregulating said functions. Examples of such sequences are promoters orsignal sequences. The DNA construct may comprise optional sequenceallowing selection of explants of Camelina sativa carrying thetransgenic inserts. The packaged combination can be provided with orwithout the media needed in the transformation procedure.

[0213] The term “assessing the efficacy of plant transformation” meansthe investigation of the rate of transgenic inclusions in transformedexplants and is assessed by recording by visible means including GUSexpression, PCR methods, Southern analysis or equivalent methods.

[0214] The term “homologous or heterologous recombinant products” meansproteins, peptides, metabolites, oils, carbohydrates, polymers, or otherproducts, which can be produced using Agrobacterium mediatedtransformation system in Camelina sativa. Homologous recombinant DNAproducts are produced when DNA sequences or genes native to Camelinasativa are used, whereas heterologous products are produced with DNAsequences or genes which are not naturally occuring in Camelina sativa.The “homologous and heterologous recombnant products” can originate frombacteria, viruses, fungi, plants and animals, including human proteinsand peptides which require processing.

GENERAL DESCRIPTION OF THE INVENTION

[0215] Brassica species have been used as common model plants in plantbreeding and molecular biology, but because they are prone to pests likeMeligethes aeneus, an alternative plant related to them would be useful.Camelina sativa would provide such a new model plant, which is notsensitive to the pest. Furthermore, Camelina sativa has a relativelysmall genome, including only 20 chromosomes, which simplifies its use ingenetic studies. Classically for example tobacco and Arabidopsis havebeen used as model plants and especially compared to Arabidopsis,Camelina sativa provides more plant material for further experiments.

[0216] In preliminary experiments we have examined different factors,which could have an effect on regeneration and transformation capacityof different explants of Camelina sativa. As a result we have developeda transformation method for plant explants, preferably leaf segments, ofCamelina sativa plants grown in vitro using Agrobacterium-mediatedtransformation.

[0217] The present invention thus provides an Agrobacterium-mediatedtransformation method of Camelina sativa.

[0218] The starting material for the transformation is Camelina sativaseed. Camelina sativa plants producing seed are grown in greenhouseconditions, since field grown plants produce seed contaminated withbacteria, which later prevents successful transformation withAgrobacterium. Camelina sativa seeds have a 0.5-1 mm thick hygroscopicpolysaccharide surface around the seed protecting the seed for exampleagainst fungal and bacterial spores and requiring more effective surfacesterilization compared to many other species. Seeds were firstoptionally sterilized and subsequently germinated and grown on Murashigeand Skoog (MS) agar medium or an equivalent plant growth medium.Preferably the leaf segments of 2-3 weeks, but preferably 10-20 days oldCamelina sativa plants preferably grown in vitro were used in theAgrobacterium-mediated transformation. Leaf segments were excised andthen placed on cultivation medium, preferebly Murashige and Skoog (MS)agar medium or an equivalent medium, supplemented with hormones, such ascytokinins and/or auxins or other hormones and sucrose or other sugarsource and cultivated on said medium for 24 hours.

[0219] The Camelina sativa explants were inoculated by immersingexplants in liquid Murashige and Skoog (MS) medium or an equivalentmedium or water containing Agrobacterium carrying the selectedtransformation vector with at least one gene foreign to said Camelinasativa.

[0220] After removing the redundant liquid on the immersed segments theexplants were placed on solid Murashige and Skoog (MS) medium or anequivalent supplemented with hormones, such as cytokinins or auxins, andoptionally with acetosyringone for co-cultivation. Explants wereco-cultivated with Agrobacterium for 2 days (48 hours) or a timesufficient to enable the transformation. During this step, theAgrobacterium transfered the foreign gene construct into Camelina sativacells. The co-cultivated segments were then washed and placed onMurashige and Skoog (MS) medium agar or equivalent medium for callus andshoot regeneration. Optional antibiotic selection begins preferablyimmediately after transformation and the result of the selection can beseen in 1-2 weeks, when explants begin to form callus. Selection wascarried out using optional antibiotics such as kanamycin, hygromycin orother selective agents for optionally 4-20 days or longer. An efficienttransformation was also obtained without selection with antibiotics.

[0221] When the selection had been completed the leaf segments had alsoproduced callus and shoots and roots. The regenerated, transgenic shootswere grown and rooted for about 2-3 weeks on Murashige and Skoog (MS)medium or equivalent medium, which is hormone-free or optionallysupplemented with auxins, including, but not limited to, indole-3-aceticacid (IAA), 4-chloro-IAA, phenylacetic acid, α-naphthaleneacetic acid(NAA) and/or 2,4-dichlorophenoxyacetic acid (2,4-D).

[0222] After rooting shoots were transferred to soil and transgenicplants were grown in greenhouse conditions (FIG. 5). Plants were testedfor GUS expression with a histological GUS assay and the presence of thetransgene was confirmed with PCR and Southern blot analysis.

[0223] The invention is also related to a system for carrying outAgrobacterium-mediated genetic transformation comprising a test kit in apackaged combination including one or more optionally sterilized seedsto provide seedlings from which explants of Camelina sativa areobtainable, an Agrobacterium vector, at least one DNA sequence encodinga desired gene product as such or in a recombinant DNA constructcomprising Agrobacterium and/or plasmids and at least one DNA sequenceencoding the desired gene product functionally combined with sequencesresponsible for or capable of regulating said functions, and optionallyat least one sequence allowing selection of explants of Camelina sativawith a culture of Agrobacterium carrying the said recombinant DNAconstruct, one or more cell culture mediums supplemented ornon-supplemented optionally with at least one hormone and/or growthfactor and/or at least one selective component which is capable ofselecting plant cells transformed with the said construct. The systemalso comprises the means and the method for obtaining whole transgenicCamelina sativa plants and growing them in vitro and in greenhouseincluding appropriate growth media, soil and equivalents.

[0224] The invention is described in more detail in the followingexperimental part. The scope of the invention is naturally notrestricted to these methods, one skilled in the art can easily replacethe suggested materials and methods with alternatives.

[0225] Materials and Methods

[0226] Plant material. The seeds of Camelina sativa were sterilized for1 min in 70% ethanol and 10 min in Na-hypochlorite (3% active Cl⁻) withaddition of Tween-20, and washed three times in sterile water. Thesterilized seeds were grown on Murashige and Skoog (MS; Murashige andSkoog, Physiol Plant. 15:472-493, 1962) agar medium or an equivalentmedium without sugars. The leaves of 2-3 weeks old, preferably 10-20days old, in vitro plants were used in the transformation experiments.

[0227] Agrobacterium vectors. Agrobacterium tumefaciens strain C58C1containing the plasmid pGV3850 (Zambryski et al., EMBO J. 2:2143-2150,1983), strain EHA105 (Hood et al., Transgenic Res. 2:208-218, 1993) withthe plasmid pTiBo542 and strain LBA4404 with pAL4404 (Hoekema et al.,Nature 303:179-180, 1983) were tested for transformation of Camelinasativa. The uidA marker gene (β-glucuronidase, GUS) containing an intron(uidA-int) (Vancanneyt et al., Mol. Gen. Genet. 220:245-250, 1990) wascloned into all vectors containing T-DNA region as listed above. TheuidA-intron-containing gene was used to prevent bacterial GUS expressionand enabled the testing of GUS-activity at an early stage oftransformation. The co-integrative pHTT294 vector, essentially similarto pHTT370 (Elomaa et al., Bio/Technology 11:508-511, 1993) carrying theuidA-intron-containing gene under the CaMV 35S promoter (Datla et al.,Plant Sci. 94:139-149, 1993), was transferred to an Agrobacterium strainC58C1. Binary pGPTV-HPT and pGPTV-KAN vectors (Becker et al., Plant.Mol. Biol. 20:1195-1197, 1992) with the uidA gene exchanged for theuidA-intron-containing gene under the control of the 35S promoter ofCaMV were transformed into Agrobacterium tumefaciens strains EHA105 andLBA4404.

[0228]Agrobacterium tumefaciens was grown overnight in liquid YEB(Lichtenstein and Draper, Genetic Engineering of Plants. In: Glover D M(ed.) DNA cloning—a practical approach, vol. 2. Oxford IRL, Oxford, pp67-119, 1985) medium with shaking supplemented with appropriateantibiotics for each strain. An aliquot ({fraction (1/100)} vol/vol) ofthe overnight culture was then inoculated in fresh YEB medium withappropriate antibiotics and bacteria were grown overnight with shaking.An Agrobacterium tumefaciens culture of OD₆₀₀=1.0 was used fortransformation.

[0229] Culture Medium

[0230] Composition of Murashige and Skoog (MS) plant growth medium:Salts: Vitamins: g/l mg/l NH₄N0₃ 1.65 Thiamine 0.1 KNO₃ ₃ 1.9 Pyridoxine0.1 MgS0₄x7H₂O 0.37 Nicotinic acid 0.5 KH₂P0₄ 0.17 Myo-inositol 100CaCl₂x2H₂0 0.44 Glycine 2.0 mg/l mg/l H₃B0₃ 6.2 Sucrose 2.0 MnSO₄x4H₂022.3 Agar 7.0 ZnSO₄x7H₂0 8.6 KJ 0.83 pH5.6 Na₂MoO₄x2H₂O 0.25 CuSO₄x5H₂O0.025 CoCl₂x2H₂0 0.025

[0231] Plant transformation. Leaf segments of in vitro grown Camelinasativa plants (FIG. 1) were cultivated for 24 hours on Murashige andSkoog (MS) medium or an equivalent medium supplemented with 0.7% agar.All MS culture media were supplemented with 2% sucrose and all in vitrocultures were kept at temperatures of 25° C. (day) and 18° C. (night)under 16 h photoperiod. Subsequently, the explants were immersed for 1-3min in Murashige and Skoog (MS) solution or an equivalent which had beeninoculated with a dilution (e.g. 1/10 vol/vol) of an overnight cultureof Agrobacterium tumefaciens. Thereafter, redundant liquid present onthe surface of leaf segments was removed using filter paper and theexplants were placed on the Murashige and Skoog (MS) agar mediumsupplemented with auxin and cytokinin hormones, 6-benzylaminopurine(BAP) and Â-naphthaleneacetic acid (NAA), for co-cultivation withbacteria for 2 days. After co-cultivation, the explants were washed withwater containing cefotaxime (Claforan) (700 mg/l), carbenicillin (200mg/l) or ticarcillin/clavulanic acid (Duchefa) (100 mg/l). The surfacesof the explants were dried on filter paper and placed on the Murashigeand Skoog (MS) medium or an equivalent medium for selection and shootregeneration. The medium was supplemented with the same hormones andantibiotics than for transgenic tissue selection (kanamycin, hygromycin)and Agrobacterium growth inhibitors.

[0232] Selection and regeneration. Eventually, cultivation of theexplants for two weeks on Murashige and Skoog (MS) medium or anequivalent medium supplemented with 0.5-1.5 mg/l 6-benzylaminopurine(BAP) and 0.1-0.5 mg/l Â-naphthaleneacetic acid (NAA) was found to bebest for callus, shoot and root formation. Thereafter, the wholeexplants or cut shoots were transferred to Petri dishes containinghormone-free culture medium, preferably Murashige and Skoog (MS) mediumor an equivalent medium, where recovered shoots elongated and started toroot. In the case that the explant forms both shoots and rootssimultaneously, whole explant is preferably transfered onto Murashigeand Skoog (MS) agar medium or an equivalent medium supplemented withcytokinins [1 mg/ml 6-benzylaminopurine (BAP)] to stimulate furthergrowth of shoots. Petri dishes were sealed with porous paper tape.Recovered transgenic shoots were grown on Murashige and Skoog (MS)medium or an equivalent medium without hormones or optionallysupplemented with 0.1-0.2 mg/l α-naphthaleneacetic acid (NAA) forstimulation of rooting, stem elongation and micropropagation. The exacthormone concentrations varied for different cultivars tested. Selectionusing hygromycin or alternatively kanamycin was applied preferablyimmediately after co-cultivation of the explants with Agrobacteriumtumefaciens. Antibiotics were used in concentrations ranging between15-25 mg/l. Selection with an antibiotic was carried out for 4-10 daysafter co-cultivation. It could be seen already after 5-7 days that theleaf segments produced callus and transgenic shoots.

[0233] Analysis of transgene expression. The histological GUS assay wasperformed on transformed callus and leaf tissue. TheuidA-intron-containing gene was used to prevent bacterial GUS expressionin transformation and to enable testing of GUS activity at early stagesof transformation, even immediately after co-cultivation withAgrobacterium tumefaciens. Usually in the optimization experiments, GUSassay was performed 4-7 days after co-cultivation with Agrobacteriumtumefaciens.

[0234] The transgenic plants which showed stable GUS expression and grewwell after selection were grown in the greenhouse (FIG. 5) They werethen used for PCR and Southern blot analysis to confirm thetransformation event at the DNA level.

[0235] Southern blot analysis was performed using a coding sequence ofuidA gene labeled with digoxigenin-11-UTP to obtain an RNA probeaccording to the manufacturer's instructions (Boehringer Mannheim).Three μg of DNA from Camelina sativa plants which showed stable GUSexpression was digested with EcoRI and BamHI restriction enzymes. Theseenzymes cut out a 2 kb uidA gene fragment from the T-region of pGPTV-HPTor pGPTV-KAN inserted in the Camelina sativa genome.

[0236] Results of the Preliminary Experiments in Developing theTransformation Method

[0237] Source plants. Field-grown Camelina sativa plants produce seedheavily contaminated and were practically improper for use in thetransformation, because leaf explants contained bacteria which preventedsuccessful transformation by Agrobacterium tumefaciens. To achieve goodstarting material seed producing Camelina sativa plants were grown ingreenhouse conditions. The seeds, which had been developed and maturedin greenhouse were free of contaminations after surface sterilization.Camelina sativa seeds have a hygroscopic polysaccharide surface, whichforms a 0.5-1 mm barrier around the seed. This barrier protects the seedagainst fungal and bacterial spores. This particular characteristics ofCamelina sativa seed surface requires more effective surfacesterilization of seeds compared to many other species. The sterilizationexperiments were performed as shown in Table 1. The Camelina sativaseeds were immersed in 70% ethanol for 1 min and treated withNa-hypochlorite solution with an addition of Tween-20 (1 drop per 100ml).

[0238] Table 1 Seed germination (%) and contamination after differentsurface sterilization treatments. Concentration of Na-hypochlorite isshown in columns as % of active Cl. The time of treatment withNa-hypochlorite is shown in rows. Time min.\% Na-hyp. 1% Cl⁻ 3% Cl⁻ 6%Cl⁻  5 min. Contaminat. 100% 60% 10 min. 100% 100% 30% 20 min. 100%  60% 0%

[0239] After sterilization the seeds were washed three times insterilized water and placed on Murashige and Skoog (MS) agar medium oran equivalent medium without sugars for germination. Germination wasassessed 3 days after sterilization. The 10 min treatment with 3%Na-hypochlorite was found optimal for Camelina sativa seedsterilization.

[0240] Sterilized seeds were germinated and grown for 2-3 weeks orpreferably 10-20 days on Murashige and Skoog (MS) agar medium or anequivalent medium without sucrose and hormones. The green leaves servedas a source for explants for the transformation.

[0241] Plant transformation. Three different Agrobacterium tumefaciensstrains, namely C58C1, EHA105 and LBA4404 were tested. C58C1pGV3850harbors the cointegrative vector pHTT294. The strains EHA105 and LBA4404carried the binary vector pGPTV-HPT. UidA-intron-containing reportergene was cloned from pGUS-int into all the binary and cointegrativevectors used in the transformation experiments. The uidA-int gene wasplaced under CaMV 35S promoter.

[0242] Hypocotyl, cotyledon, leaf and stem segments were tested foraffinity to Agrobacterium tumefaciens. The cotyledon and leaf segmentshad the best transformation capacity. Because of their betterregeneration ability, leaf segments were used in further transformationexperiments. Leaves of in vitro grown Camelina sativa plants are rathersmall in size: 2 to 4 cm long and 0.5-1 cm wide. Therefore, narrowleaves were cut only across the leaf while larger leaves were also cutin half along the central vein.

[0243] Transformation efficiencies of different Agrobacteriumtumefaciens strains were measured as a proportion of blue inclusions incallus one week after inoculation of leaf segments (FIGS. 3a, 3 b).

[0244] Table 2 Transformation efficiencies of different Agrobacteriumtumefaciens strains. First column: GUS positives/all explants, Secondcolumn: intensive transformation. Blue inclusions IntensivelyTransformation Agrobacterium all explants transformed % (intensive)LBA4404pGPTV-HPT 35/50 7/50 70% (14) EHA105pGPTV-HPT 24/50 48% (0)C58C1pGV3850 pHTT294 33/50 4/50 66% (8)

[0245] The results of the three transformation experiments, summarizedin Table 2, showed that LBA4404 and C58C1pGV3850 strains were effectivein transforming Camelina sativa. EHA105 was slightly less effective. Theexplants infected with LBA4404 or C58C1 strains had large intensivelystained blue inclusions. Thus, the strains LBA4404 and C58C1 were usedin subsequent transformation experiments.

[0246] Shoot regeneration. Effects of different hormones on variousexplants of Camelina sativa (hypocotyl, cotyledon, leaf and stemsegments) were tested in preliminary experiments to achieve sufficientshoot regeneration. 6-benzylaminopurine (BAP) and α-naphthaleneaceticacid (NAA) were more effective to induce shoot and root regenerationthan kinetin and indole-3-acetic acid (IAA). The regeneration capacityof cotyledons was 30-50% whereas shoots from hypocotyl and stem segmentsdid not regenerate. The best regeneration (100%) was achieved with leafsegments (FIG. 2). The 2,4-dichlorophenoxyacetic acid (2,4-D),gibberellins as well as silver nitrate treatments did not have an effecton shoot regeneration. The best regeneration was achieved with a certainratio of auxin and cytokinin hormones. For example, the best shootregeneration of leaf segments of Camelina sativa variety cv. Calena wasachieved with the hormone combination of 1 mg/l 6-benzylaminopurine(BAP) and 0.2 mg/l α-naphthaleneacetic acid (NAA), while the optimalcombination for Camelina sativa variety cv. Calinca was 0.7 mg/l6-benzylaminopurine (BAP) and 0.3 mg/l α-naphthaleneacetic acid (NAA).

[0247] Recovered shoots had a tendency for inflorescence formation andhad problems with rooting. To overcome these problems, recovered shootswere cultivated subsequently on Murashige and Skoog (MS) medium or anequivalent medium optionally supplemented with auxins (e.g.indole-3-acetic acid (IAA) 1 mg/l). Another way was to regenerate shootsand roots simultaneously with the hormone combination of 0.5-1 mg/l6-benzylaminopurine (BAP) and 0.2-0.7 mg/l α-naphthaleneacetic acid(NAA).

[0248] Several different factors were tested for impact on shootregeneration efficiency. Optimal parameters were found for pH (5.6-5.8),for sucrose content (2-3%), and solidifiers (0.7% agar). Modificationsin the concentration of NH₄, NO₃ ⁻, K⁺ and Ca²⁺ ions in the standardMurashige and Skoog (MS) medium had no effect nor did the addition ofglucose. Culturing the explants on the B5 medium had also no effect onshoot regeneration.

[0249] Selection. To prevent Agrobacterium tumefaciens growth on themedium, cefotaxime (Claforan) (500 mg/l), carbenicillin (200 mg/l),ticarcillin/clavulonic acid (Duchefa) (100 mg/ml) or vancomycin (200mg/ml) were used.

[0250] The selection markers i.e. the hpt and nptII genes intransformation constructs provided the plants with resistance tohygromycin and kanamycin, respectively. It had been found that theapplication of a selection pressure (15-20 mg/l, preferably 10-20 mg/lof antibiotic) preferably for 4-10 days after washing of theAgrobacterium tumefaciens from explants is optimal. First regenerativeprimordia form on the calli already 10 days after cutting of the leafsegments, and selection of transformed tissues should be performedbefore that. It was found in preliminary experiments that 5-15 mg/lantibiotic prevented morphogenesis of explants. Selection of transformedtissue using 5-10 mg/l hygromycin or kanamycin was not enough. On theother hand, the concentrations of the antibiotic higher than 20-30 mg/lkilled the explants too fast for any shoots to recover.

[0251] Analysis of Transformation

[0252] The histological GUS assay was performed on transformed callusand leaf tissue. To prevent GUS expression in Agrobacterium tumefaciens,the uidA gene containing the intron was used in transformationexperiments. It enabled the testing of GUS activity almost immediatelyafter co-cultivation with Agrobacterium tumefaciens. Usually, GUS assaywas made 4-7 days after co-cultivation with Agrobacterium tumefaciensduring the optimization of transformation (FIG. 3). The assay was alsoperformed on regenerated primordia and shoots as well as leaf segmentsof recovered plants.

EXAMPLE 1

[0253] Transformation Protocol for Camelina sativa cv. Calena withAgrobacterium tumefaciens Strain LBA4404 Harboring the Binary PlasmidpGPTV-HPT with uidA Intron Containing Gene.

[0254] The seeds of Camelina sativa plant grown in greenhouse weresterilized by immersing in 70% ethanol for 1 min and then treating for10 min with Na-hypochlorite solution (3% active Cl⁻) with an addition ofTween-20 (1 drop per 100 ml). After sterilization the seeds were washedthree times in sterile water and placed on solid Murashige and Skoog(MS) agar medium (Murashige and Skoog, Physiol. Plant. 15:472-493, 1962)without sugars for germination. Sterilized seeds were germinated andgrown 2-3 weeks on solid Murashige and Skoog (MS) medium withouthormones (FIG. 1). Green leaves served as a source of explants fortransformation procedure.

[0255]Agrobacterium tumefaciens strain LBA4404 carrying pGPTV-HPT-GUSintvector was grown overnight at 28° C. with shaking in liquid YEB medium(Lichtenstein and Draper, Gene Engineering of Plants. In: Glover D M(ed.) DNA Cloning—A Practical Approach, vol. 2. Oxford IRL, Oxford, pp67-119, 1985) supplemented with 50 mg/l kanamycin and rifampicin.Subsequently an aliquot of the culture ({fraction (1/100)} v/v) wasinoculated in fresh YEB medium supplemented with 50 mg/l kanamycin andrifampicin and the bacteria were grown overnight with shaking.Agrobacterium culture of OD₆₀₀=1.0 was used in the transformationexperiments.

[0256] Narrow leaves of in vitro grown Camelina sativa plants were cutonly across the leaf blade, whereas large leaves were additionally cutin half along the central vein. The leaf segments were cultivated for 24hours on Murashige and Skoog (MS) 0.7% agar medium supplemented with 1mg/l 6-benzylaminopurine (BAP) and 0.2 mg/l α-naphthaleneacetic acid(NAA). All the Murashige and Skoog (MS) culture media were supplementedwith 2% sucrose and all in vitro cultures were kept at temperatures of25° C. (day) and 18° C. (night) under the photoperiod of 16 h. Theexplants were immersed for 1-3 min in Murashige and Skoog (MS) solutioninoculated with a dilution (e.g. {fraction (1/10)} v/v) of the overnightculture of Agrobacterium tumefaciens LBA4404. Redundant liquid on thestem segments was removed using filter paper and the explants wereplaced on Murashige and Skoog (MS) agar medium supplemented with auxinand cytokinin for co-cultivation with bacteria for 2 days. The explantswere washed with water containing claforan [cefotaxime) (700 mg/l)] orcarbenicillin (700 mg/ml). The surfaces of the explants were dried onfilter paper and the explants were placed on Murashige and Skoog (MS)medium supplemented with hormones [0.7 mg/l 6-benzylaminopurine (BAP),0.25 mg/l α-naphthaleneacetic acid (NAA)] and 200 mg/l carbenicillin orclaforan and 15 mg/ml hygromycin. Two to three weeks old shoots (FIG. 2)were placed to the normal or half strength Murasghige and Skoog (MS)medium solidified with 0.7% agar and supplemented with 200 mg/lcarbenicillin or cefotaxime and optionally with 15 mg/l hygromycin andauxin [indole-3-acetic acid (IAA) 0.5-1 mg/l] shoots were transferred tosoil and transgenic plants were grown in greenhouse conditions (FIG. 5).Transgenic plants were tested for uidA (GUS) gene expression with ahistological GUS assay and the presence of the transgene was confirmedwith Southern analysis.

EXAMPLE 2

[0257] Transformation Protocol for Camelina sativa cv. Calinca withAgrobacterium tumefaciens Strain C58C1 pGV3850 Harboring the Binary TiVector with Kanamycin Selection.

[0258] Seeds were taken from greenhouse grown Camelina sativa cv.Calinca plants (no older than 4 months). Transformation efficiencyincreases from 66% to 100% if donor plants are grown in greenhouse.

[0259] 10 Days Before Excision of the Explants.

[0260] Seeds of Camelina sativa were sterilized and placed in vitro onMurashige and Skoog (MS) agar medium without sucrose and grown attemperatures of 25° C. (day) and 18° C. (night) as described in Example1.

[0261] 1^(st) Day.

[0262] A fresh colony of Agrobacterium tumefaciens strain C58C1pGV3850carrying binary pGPTV-KAN vector (Becker et al., Plant Mol. Biol.20:1195-1197, 1992) containing uidA-int gene under 35S promoter andselectable marker gene nptII, was inoculated in 3 ml of liquid YEBmedium supplemented with 25 mg/l rifampicin (Rif) and 50 mg/l kanamycin(Kan). The bacteria were grown overnight with shaking at 28° C.

[0263] 2^(d) Day. Pre-Cultivation.

[0264] The first leaves (no cotyledons) of in vitro grown Camelinasativa were cut into segments across the leaf and were placed onpre-cultivation plates containing 0.7% MS agar medium supplemented with2% sucrose, 0.7 mg/l 6-benzylaminopurine (BAP) and 0.3 mg/lÂ-naphthaleneacetic acid (NAA). All dishes were sealed with porous papertape (Micropore 3M). A 30 μl aliquot of overnight culture of theAgrobacterium tumefaciens was inoculated in 3 ml of fresh YEB mediumsupplemented with rifampicin (Rif) and kanamycin (Kan).

[0265] 3^(d) Day. Agrobacterium tumefaciens Inoculation.

[0266] The explants were immersed in liquid Murashige and Skoog (MS)medium supplemented with 2% sucrose and inoculated with a {fraction(1/10)} (v/v) dilution of the overnight culture of Agrobacteriumtumefaciens. After 5 min inoculation redundant liquid on the explantswas removed using sterilized filter paper.

[0267] The explants were placed on Murashige and Skoog (MS) mediumsupplemented with 2% sucrose for co-cultivation with the Agrobacteriumtumefaciens for two days at 28° C. in dim light.

[0268] 5^(th) Day. Washing and Selection.

[0269] The explants were washed with water containing 100 mg/lticarcillin/clavulanic acid (Duchefa). Ticarcillin (Tc) has lessnegative effect on shoot and root regeneration than cefotaxime(Claforan) and carbenicillin. On the other hand it is more effectivegrowth inhibitor of Agrobacterium tumefaciens than vancomycin. Theexplants were dried on the filter paper and transferred onto selectionmedium containing 0.7% Murashige and Skoog (MS) agar medium supplementedwith 2% sucrose, 0.7 mg/l 6-benzylaminopurine (BAP), 0.3 mg/lα-naphthaleneacetic acid (NAA), 15 mg/l kanamycin and 50 mg/lticarcillin/clavulanic acid (Duchefa). The explants were cultured on theselection medium for 4-5 days.

[0270] 10^(th) Day. Regeneration.

[0271] The explants were transferred onto plates containing 0.7% MS agarmedium supplemented with 2% sucrose, 0.7 mg/l 6-benzylaminopurine (BAP),0.3 mg/l α-naphthaleneacetic acid (NAA), and 50 mg/lticarcillin/clavulanic acid (Duchefa) for shoot and root regenerationfor 10-14 days. Tall (3 cm high) plates were sealed with porous papertape to increase aeration. Simultaneous regeneration of shoots and rootsis preferable for effective recovery of transgenic Camelina sativaplants.

[0272] 20-24^(th) Day. Shoot and Root Elongation.

[0273] Explants forming 0.5-1 cm long leaves (shoots) and roots weretransferred on 0.7% Murashige and Skoog (MS) agar medium containing 2-3%sucrose and 100 mg/l ticarcillin/clavulanic acid without hormones oroptionally supplemented with 1 mg/ml 6-benzylaminopurine (BAP) for 5-7days. Alternatively explants were transferred to fog system (mistchamber) in greenhouse for consecutive growth.

[0274] 25-30^(th) Day. Transgenic Plant Growth.

[0275] Successfully grown and rooted shoots were transferred to soilwithout separation from explants. Shoots in pots were placed into closedchamber. The chamber was opened gradually day by day to increaseaeration. Alternatively explants were transferred to fog system (mistchamber) in greenhouse for consecutive growth. The recovered shootsformed inflorescence and seedpods. Plant tissues were tested forexpression of marker gene (GUS) with GUS assay, PCR and Southern blot.

EXAMPLE 3

[0276] Transformation Protocol for Camelina sativa cv. Calena withAgrobacterium tumefaciens Strain 4 C58C1 pGV3850 Harboring CointegrativeTi DNA Without Selection of Transgenic Tissues.

[0277] Seeds were taken from greenhouse grown Camelina sativa cv. Calenaplants (no older than 4 months). Transformation efficiency increasesfrom 66% to 100% if donor plants are grown in greenhouse.

[0278] 10 Days Before Explants Excision.

[0279] Seeds were sterilized and placed in vitro on Murashige and Skoog(MS) medium without sucrose and grown at temperatures of 25° C. (day)and 18° C. (night) as described in Example 1.

[0280] 1^(st) Day.

[0281] A fresh colony of C58C1pGV3850 with interned Ti DNA from pHTT-HPT(Elomaa et al., Bio/Technology 11:508-511, 1993) vector containing GUSgene under 35S promoter and hpt selectable marker was inoculated in 3 mlof liquid YEB supplemented with 25 mg/l rifampicin (Rif) and 100 mg/lspectinomycin (Spe) or streptomycin (Str). The bacteria were grownovernight with shaking at 28° C.

[0282] 2^(nd) Day. Pre-Cultivation.

[0283] The first leaves (no cotyledons) were cut into segments acrossthe leaf and placed onto the pre-cultivation plates containing 0.7%Murashige and Skoog (MS) agar medium with 2% sucrose supplemented with 1mg/l 6-benzylaminopurine (BAP) and 0.5 mg/l Â-naphthaleneacetic acid(NAA). All plates were sealed with porous paper tape (Micropore 3M).

[0284] A 30 μl aliquot of overnight culture of the Agrobacteriumtumefaciens was inoculated in 3 ml of fresh YEB medium supplemented withrifampicin (Rif), spectinomycin (Spe) or streptomycin (Str).

[0285] 3^(rd) Day. Agrobacterium Inoculation.

[0286] The plant explants were immersed in liquid Murashige and Skoog(MS) medium supplemented with 2% sucrose and inoculated with a {fraction(1/10)} dilution of the overnight culture of Agrobacterium tumefaciens.Redundant liquid on the explants was removed on sterilized filter paper.The explants were co-cultivated with the Agrobacterium tumefaciens fortwo days at 28° C. in dim light.

[0287] 5^(th) Day. Washing and Reegneration.

[0288] The explants were washed with water containing 100 mg/lticarcillin/clavulanic acid (Duchefa). Ticarcillin (Tc) has lessnegative effect on shoot and root regeneration compared to cefotaxime(Claforan) and carbenicillin. On the other hand it is more effectivegrowth inhibitor of Agrobacterium tumefaciens than vancomycin. Theexplants were dried on the filter paper. Then the explants were placedonto selection medium plates containing 7% Murashige and Skoog (MS) agarmedium with 2% sucrose supplemented with 1 mg/l 6-benzylaminopurine(BAP), 0.5 mg/l α-naphthaleneacetic acid (NAA) and 50 mg/lticarcillin/clavulanic acid (Duchefa) 0.5 mg/l for shoot and rootregeneration for 2-3 weeks. Tall (3 cm high) plates were sealed withporous paper tape to increase aeration.

[0289] 20-24^(th) Day. Shoot and Root Elongation.

[0290] Explants forming 0.5-1 cm long leaves (shoots) and roots weretransferred onto 0.7% Murashige and Skoog (MS) agar medium containing 2%sucrose supplemented with 100 mg/l ticarcillin/clavulanic acid (Duchefa)without hormones or with 1 mg/ml 6-benzylaminopurine (BAP) for 5-7 days.Plates were not sealed with tape.

[0291] Regenerated shoots were tested for GUS expression withhistological GUS assay. GUS activity was seen in 4 shoots out of 123. Itmeans that average of about 3% of shoots regenerated aftertransformation were transgenic without the use of antibiotic selection.Thus the method can be used for producing transgenic Camelina sativaplants free from antibiotic resistance genes or selectable marker genes.

[0292] The strain C58C1pGV3850 was the most effective for transformationof Camelina sativa. 100% of the explants were transformed. The averageproportion of tissue in each explant showing GUS expression was morethan 30%. This is the highest level of transformation that wasregistered by present inventors. The transformation efficiency enablesto obtain transgenic plats without antibiotic or other selection oftransgenic plants.

EXAMPLE 4

[0293] Analysis of Transformation.

[0294] The histological GUS assay was performed on transformed callusand leaf tissue. To prevent GUS expression in Agrobacteria the uidA genecontaining an intron was used in transformation experiments. It enabledthe testing of GUS activity even immediately after co-cultivation withAgrobacterium tumefaciens. Usually, GUS assay was made 4-7 days afterco-cultivation with Agrobacterium tumefaciens during the optimization oftransformation (FIG. 3). The assay was also performed on regeneratedprimordia and shoots as well as leaf segments of recovered plants.

[0295] Transgenic plants which showed steady positive GUS expression andgrew well under selection conditions were used for PCR analysis oftransgene insertion and Southern blot analysis to confirm thetransformation events.

[0296] PCR Analysis.

[0297] Total genomic DNA was isolated from leaf tissue of transgenic andnon-transgenic Camelina sativa plants using DNeasy Plant Mini Kitaccording to the supplier's instructions (Qiagen). The presence of theuidA and hpt gene in the GUS positive plants was determined by PCRanalysis using 24 nucleotides long primers specific to the codingsequences of uidA and hpt genes. PCR reaction mix containedapproximately 1 ng/μl of template DNA and DyNAzyme polymerase(Finnzymes) was used for amplification. PCR program conisted of: 94° for2 min; 30 cycles of 94° C. for 30 sec, 48° C. for 30 sec and 72° C. for2 min. Three μl of PCR reaction mixture was run in 0.8% agarose gelcontainig ethidium bromide at 100 V. No PCR product was obtained whennon-transgenic Camelina sativa DNA was used as template, whereas whenusing transgenic Camelina sativa an amplification product of 700nucleotides corresponding to the positive control was obtained whichconfirmed the presence of transgene in transgenic Camelina sativa plants(FIG. 4).

[0298] Southern Analysis

[0299] Total genomic DNA was isolated from leaf tissue of Camelinasativa plants using DNeasy Plant Midi Kit according to the supplier'sinstructions (Qiagen). Three μg of DNA from GUS positive Camelina sativaplants was digested with EcoRI and BamHI restriction enzymes. Theseenzymes cut out a 2 kb uidA gene fragment from the T-region of pGPTV-KAN(-HPT) inserted in the plant genome. Digested DNA samples were separatedin a 0.7% agarose (Promega) gel overnight at 15 mA current andtransferred to positively charged nylon membrane (Boehringer Mannheim)using vacuum blotter. RNA probes were synthesized using T3 RNApolymerase on the pBluescript vector carrying uidA or hpt gene sequenceand labeled with digoxigenin-11-UTP. The membrane was hybridized anddeveloped according to the supplier's instructions (Boehringer Mannheim,The DIG user's guide for filter hybridization). The membrane wasprehybridized at 50° C. for 2 h and hybridized at 50° C. in a “DIG EasyHyb” hybridization solution (Boehringer Mannheim) overnight with adigoxigenin-UTP labeled RNA probe. The concentration of RNA probe was100 ng/ml. After hybridization the membrane was washed in SSC buffers,blocked and detected using “Anti-Digoxigenin-AP alkaline phosphatase(Boehringer Mannheim). Chemiluminescent detection was done with withCSPD-substrate and the membrane was exposed to X-ray film (BoehringerMannheim). Presence of the transgene insertion was proved in comparisonto DNA of non-transgenic Camelina sativa plant DNA as negative control,and to plasmid DNA carrying the gene sequence mixed with non-transgenicplant DNA as positive control.

1. A method for Agrobacterium-mediated genetic transformation,characterized in that the method is Agrobacterium-mediated genetictransformation of Camelina sativa comprising the steps of: (a) providingexplants from Camelina sativa; (b) contacting the explants of Camelinasativa with Agrobacterium containing at least one recombinant DNAconstruct; (c) allowing the transformation to take place on culturemedium optionally supplemented with at least one hormone; (d) inducingformation (regeneration) of one or more shoots and roots from thetransformed explants on a cell culture medium optionally containing atleast one hormone; and (e) growing the shoots into a whole Camelinasativa plant.
 2. The method according to claim 1, characterized in thatthe explants are obtained from Camelina sativa plants grown fromoptionally sterilized seeds.
 3. The method according to claim 1,characterized in that the Agrobacterium is Agrobacterium tumefaciens. 4.The method according to claim 1, characterized in that the recombinantDNA construct is a vector comprising an optional selectable marker gene.5. The method according to claim 1, characterized in that transformedtissue of Camelina sativa is optionally selected on a medium containinga selective component.
 6. The method according to claim 1, characterizedin that the Camelina sativa explant is selected from hypocotyl,cotyledon, stem, leaf or other plant organs.
 7. The method according toclaim 1, characterized in that the selectable marker gene comprisesantibiotic resistance genes or mixtures therof.
 8. The method accordingto claim 7, characterized in that the antibiotic resistance genecomprises neomycin phosphotransferase (npt II) gene or hygromycinphosphotransferase (hpt) gene or mixtures thereof.
 9. The methodaccording to claim 1, characterized in that the selective component isantibiotic.
 10. The method according to claim 9, characterized in thatthe antibiotic is hygromycin or kanamycin.
 11. The method according toclaim 1, characterized in that the induction of the shoots and rootscomprises application of hormone or a growth factor allowingregeneration.
 12. The method according to claim 1, characterized in thatthe recombinant DNA construct is a DNA sequence comprising at least onegene of interest, at least one promoter sequence and an optionalselectable marker gene.
 13. The method according to claim 1,characterized in that the optional hormone is cytokinin, auxin,gibberellin or mixtures thereof.
 14. The method according to claim 13,characterized in that the cytokinin comprises 6-benzylaminopurine (BAP),kinetin, zeatin, zeatin riboside, dihydrozeatin, isopentenyl adenine ormixtures thereof.
 15. The method according to claim 13, characterized inthat the auxin comprises Â-naphthaleneacetic acid (NAA), indole-3-aceticacid (IAA), 4-chloro-IAA, phenylacetic acid, 2,4-dichlorophenoxyaceticacid (2,4-D) or mixtures thereof.
 16. The method according to claims1-15 for obtaining a transgenic Camelina sativa plant.
 17. The methodaccording to claims 1-15 for obtaining transgenic Camelina sativa planttissue.
 18. The method according to claims 1-15 for obtaining transgenicCamelina sativa plant cells and cell lines.
 19. A transgenic plant,characterized in that it comprises a Camelina sativa plant obtainablewith the method of claim
 1. 20. A transgenic plant tissue, characterizedin that it comprises Camelina sativa tissue obtainable with the methodof claim
 1. 21. A transgenic plant cell or cell lines, characterized inthat it comprises Camelina sativa cells or cell lines obtainable withthe method of claim
 1. 22. Transgenic seed, characterized in that itcomprises Camelina sativa seed obtainable with the method of claim 1.23. The use of the method according to any of claims 1-15 for providinga transformation system in Camelina sativa.
 24. The use of the methodaccording to any of claims 1-15 to produce homologous or heterologousrecombinant products in Camelina sativa.
 25. The use of the methodaccording to claim 24 where the homologous or heterologous products areproteins of metabolites.
 26. The use of the method according to any ofclaims 1-15 for producing oil products with unique properties inCamelina sativa.
 27. The use of Camelina sativa as an alternative modelplant in Agrobacterium-mediated transformation.